Method of creating human pluripotent stem cell derived brain pericyte-like cells

ABSTRACT

A population of brain pericyte-like cells, wherein the cells express pericyte markers but do not express ACTA2 and wherein the cells are generated from hPSCs, is disclosed herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.16/507,586, filed Jul. 10, 2019, which claims priority to U.S.Provisional Application No. 62/696,230 filed on Jul. 10, 2018, thecontents of which are incorporated by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under HDTRA1-15-1-0012awarded by the DOD/DTRA and NS083688 awarded by the National Institutesof Health. The government has certain rights in the invention.

REFERENCE TO AN ELECTONIC SEQUENCE LISTING

The contents of the electronic sequence listing (960296_02506_Sequencelisting.xml; Size: 46,006 bytes; and Date of Creation: May 9, 2023) isherein incorporated by reference in its entirety.

BACKGROUND

The blood-brain barrier (BBB) is comprised of specialized brainmicrovascular endothelial cells (BMECs) that line the vasculature of thecentral nervous system (CNS). BMECs allow for the selective passage ofessential nutrients and metabolites into the brain and help prevent theentry of damaging substances. While the BBB plays an important role inCNS homeostasis, it also creates a bottleneck for the delivery oftherapeutics¹⁻³. In addition, BBB dysfunction has been observed in manyCNS pathologies including Alzheimer's disease, multiple sclerosis, andstroke, and increasing evidence demonstrates treating BBB contributionto CNS disorders may improve disease outcomes⁴⁻¹². Importantly, BMECsgain their unique properties as a result of coordinated signaling cuesfrom other brain cells surrounding CNS microvessels, including CNSpericytes, astrocytes, and neurons that together with BMECs form theneurovascular unit (NVU)¹³⁻¹⁸. Recently, brain pericyte contributions toBBB development and function have begun to be elucidated, and potentialpericyte roles in CNS disease have been suggested. CNS pericytesassociate with BMECs early in embryonic development as nascent bloodvessels invade the developing neural tube. The emergence of pericytescorresponds to BBB formation through reduction of transcytosis,decreased immune cell adhesion molecule expression, and reducedultrastructural tight junction abnormalities¹³. In the adult, pericytesregulate vascular stability and diameter^(5,19-21), contribute to theBMEC basement membrane^(20,22-24), regulate BMEC molecularphenotype^(14,25), and reduce non-specific molecular transcytosis¹⁴.

As a result of the emerging importance of brain pericytes in brainhealth and disease, they have been increasingly incorporated into invitro models of the BBB. For example, co-culture with pericytes canimprove BMEC phenotype in co-culture systems, stabilize endothelial cellcord formation in vitro²⁶, and induce BMEC properties in primary andhematopoietic stem cell-derived endothelial cells²⁷⁻²⁹. We also reportedthat primary brain pericytes could be combined with human pluripotentstem cell derived BMECs (hPSC-derived BMECs) and enhance theirfunctionality³⁰. Such hPSC-derived BBB models offer the capability forscreening of CNS-penetrant therapeutics³¹ and can be used to investigateBBB contributions to human disease using patient-derived inducedpluripotent stem cells (iPSCs)^(32,33). While we and others haverecently demonstrated the combination of iPSC-derived BMECs withiPSC-derived astrocytes and neurons to form high fidelity multicellularBBB mode³⁴⁻³⁶, the inclusion of pericytes, to date, has largely beenlimited to primary human sources^(30,35). Unfortunately, primary sourcesdo not scale with high fidelity^(37,38), and unlike iPSC sources, do notreflect the genetic contributions that can be important to modelinghuman disease. Thus, for patient-specific modeling of the healthy anddiseased BBB, it is paramount to generate brain pericyte-like cells fromhuman iPSCs.

Vascular mural cells include both smooth muscle cells, which linearterioles and venules, and pericytes, which are associated with smallermicrovessels and capillaries. Until very recently, it has been difficultto distinguish smooth muscle cells from pericytes based on markerexpression³⁹. Moreover, hPSC-derived mural cells from differentembryonic origins display functionally distinct phenotypes and responddifferentially to disease pathways^(40,41). While most mural cellsoriginate from mesoderm, CNS forebrain mural cells arise from neuralcrest stem cells (NCSCs)^(42,43), a multipotent stem cell populationcapable of forming peripheral neurons and mesenchymal derivativesincluding adipocytes, osteocytes, and chondrocytes^(44,45), among othercell types. Previous studies have described processes to differentiatehPSCs to NCSCs and demonstrated their potential to form vascular smoothmuscle cells^(41,45,46). However, it is unknown whether NCSCs cangenerate pericyte-like cells that enhance BBB phenotypes in BMECs. Here,we describe a facile protocol for generating multipotent NCSCs fromhPSCs by canonical WNT signaling activation with simultaneous inhibitionof BMP and activin/nodal signaling as previously described^(45,47).These hPSC-derived NCSCs can be further differentiated to mural cellsthat express pericyte markers by 9 days of culture in serum-containingmedium. These pericyte-like cells associated with vascular cord networksand induced key pericyte-driven phenotypes in BMECs including theenhancement of barrier properties and reduction of transcytosis.Finally, an isogenic model of the NVU comprised of iPSC-derivedpericytes, BMECs, astrocytes, and neurons, exhibited elevated barrierproperties compared to a model lacking pericytes, suggesting futureapplications of iPSC-derived pericytes in CNS drug screening, BBBdevelopment studies and disease modeling applications.

Needed in the art is an improved method of creating iPSC-derivedpericytes.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a population of brainpericyte-like cells, wherein the cells express pericyte markers but donot express ACTA2 and wherein the cells are generated from hPSCs.Further aspects provide methods of producing such cells. In one aspect,the suitable pericyte markers include CNN1, NG2, and PDGFRB. Furtheraspects comprise blood brain barrier models comprising the brainpericyte-like cells.

In another aspect, the disclosure provides a method of creating apopulation of brain pericyte-like cells, wherein the cells expresspericyte markers but do not express ACTA2 and wherein the cells aregenerated from human pluripotent stem cells (hPSC), comprising the stepsof a. culturing hPSC in E6-CSFD medium for about 15 days to producedp75-NGFR+HNK+NCSC cells, b. sorting p75-NGFR⁺ cells and re-plating thep75-NGFR⁺ cells to produce an enriched population of p75-NGFR⁺NCSCs, andc. culturing the cells of step (b) in E6 media with an addition of serumfor about 11 days, wherein a brain pericyte-like population of cellsthat express pericyte markers but do not express ACTA2 is produced.

In another aspect, the disclosure provides a method of creating apopulation of p75-NGFR+HNK+NSCs from human pluripotent stem cells, themethod comprising: a. culturing hPSC in E6-CSFD medium for about 15 daysto produced p75-NGFR+HNK+NCSC cells, and b. sorting p75-NGFR⁺ cells andre-plating the p75-NGFR⁺ cells of step (a) to produce a population ofp75-NGFR⁺ NCSCs.

BRIEF DESCRIPTION OF DRAWINGS

The patent or patent application file contains at least one drawing incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1J. Generation of multipotent NCSC populations. A) NC SCdifferentiation timeline. Small molecule activation of canonical Wntsignaling and small molecule inhibition of Activin/Nodal/TGFβ/BMPsignaling in minimal medium produces H9-derived NCSCs over a 15 daytreatment window. NCSCs are then magnetically sorted and replated forsubsequent mural cell differentiation. B) Immunocytochemistry images ofH9 hESCs differentiated in E6-CSFD probed for the presence of HNK1 andp75-NGFR at D15. NCSCs are HNK1⁺/p75-NGFR⁺ cells. Hoechst nuclearcounter stain (blue) is also included. Scale bar: 100 μm. C) AP-2immunocytochemistry images for H9-derived NCSCs at D15. Hoechst nuclearcounter stain (blue) is also included. Scale bar: 100 μm. D) TemporalPCR analysis of pluripotency (NANOG, POU5F1) and NCSC (TFAP2A, B3GAT1,NGFR, SOX9, SOX10) transcripts. E) Quantification of NC SC expansion inpopulation doublings over the 15 days of NCSC differentiation. Plottedare the mean±SD of three technical replicates of a representativedifferentiation. F) Flow cytometry analysis of H9-derived NCSCs. Panelsinclude isotype controls (panel i), NCSC (HNK1⁺/p75-NGFR⁺) purity priorto MACS (panel ii), and NCSC purity following MACS (panel iii). Insetpercentages are included in each quadrant. Quantitation is shown in FIG.1J. G) Immunocytochemistry analysis of D16 NCSCs following MACS andreplating. NCSCs maintained HNK1 and p75-NGFR expression. Hoechstnuclear counter stain (blue) is also included. Scale bar: 100 μm. H)Immunocytochemistry analysis of H9-derived NCSCs subsequentlydifferentiated in peripheral neuron medium. Resultant cells werepositive for βIII-tubulin and peripherin expression. Hoechst nuclearcounter stain (blue) is also included. Scale bar: 200 μm. I) H9-derivedNCSCs could be differentiated into mesenchymal derivatives, includingOil Red O stained adipocytes (panel i, red), Alizarin red stainedosteocytes (panel ii, red), and Alcian blue stained chondrocytes (panelii, blue). Scale bar: 200 μm. J) NC SC and pericyte-like celldifferentiation efficiencies. J) NCSC and pericyte-like celldifferentiation efficiencies for three hPSC lines.

FIGS. 2A-2J. Serum treatment directs H9-derived NCSCs towards muralcells. A) Differentiation timeline for mural cell differentiation.Replated NCSCs are differentiated to mural cells in E6 medium plus 10%FBS for 9 days. B) PDGFRβ and NG2 immunocytochemistry of cells obtainedafter treating replated H9-derived NCSCs for 6 days in E6,E6+TGFβ1+PDGF-BB, or E6+10% FBS on uncoated tissue culture polystyrene,or E6+10% FBS on gelatin-coated tissue culture polystyrene. C) Temporalflow cytometry analysis for PDGFRβ and NG2 positive cells in H9-derivedNCSCs treated with E6+10% FBS. Depicted are the means±SEM of at leasttwo independent differentiations at each time point, *P<0.05 vs. D15NCSC using ANOVA followed by Dunnett's test. D) Representative PDGFRβand NG2 flow cytometry plots for H9-derived NCSC treated 9 days withE6+10% FBS medium. Quantitative data can be found in FIG. 1J. E)Temporal PCR analysis of mural and pericyte transcripts for thedifferentiating H9 hESCs. F) PDGFRβ and NG2 immunocytochemistry ofH9-derived NCSCs (D16), mural cells (D22), and primary pericytes.Hoechst nuclear counter stain (blue) is also included. Scale bar: 200μm. G) Calponin and SM22α immunocytochemistry of H9-derived NCSCs (D16),mural cells (D22) and primary pericytes. Hoechst nuclear counter stain(blue) is also included. Scale bar: 200 μm. H) α-SMA immunocytochemistryof H9-derived NCSCs (D16), mural cells (D22) and primary pericytes.Hoechst nuclear counter stain (blue) is also included. Scale bar: 200μm. I) CD13 immunocytochemistry of H9-derived mural cells (D22). Hoechstnuclear counter stain (blue) is also included. Scale bar: 200 μm. J)Desmin immunocytochemistry of H9-derived mural cells (D22). Hoechstnuclear counter stain (blue) is also included. Scale bar: 200 μm.

FIGS. 3A-3D. RNA-sequencing of pericyte-like cells and related celltypes. A) Hierarchical clustering based on all transcripts ofundifferentiated H9 hESCs, H9-derived NCSCs at D15 and after anadditional 40 days in E6-CSFD (D55), H9-derived pericyte-like cells atD19, D22, and D25 (three independent differentiations at the D25 timepoint, indicated as “H9-A”, “H9-B”, and “H9-C”), H9-derivedpericyte-like cells maintained for an additional 20 days in E6+10% FBS(D45), CS03n2- and IMR90C4-derived pericyte-like cells at D25, andprimary brain pericytes (from two distinct cultures of the same cellsource, indicated as “Primary-A” and “Primary-B”). B) Expression (FPKM)of selected transcripts in H9 hPSCs (day “0”), NCSCs (“15”), and duringthe differentiation of pericyte-like cells (“19”, “22”, “25”, and “45”).Also shown is the mean transcript expression in all D25 hPSC-derivedpericyte-like cells (H9 A-C, CS03n2 and IMR90C4, “H”) and in primarybrain pericytes (“P”). Error bars represent SEM of five independentdifferentiations (“H”) or of two primary pericyte samples (“P”). C) Top10 gene ontology (GO) terms, sorted by enrichment score(ES=−log₁₀(FDR)), for hPSC-derived pericyte-like cells. Genes includedin the dataset were enriched in pericyte-like cells (average of all D25samples) compared to NCSCs (average of D15 and D55 samples)(FPKM_(pericyte-like cells)/FPKM_(NCSC)≥10), and were expressed at ≥1FPKM in pericyte-like cells. D) Expression (≥1 FPKM) of murinepericyte-enriched transcripts (46 transcripts (40)) in hPSC-derivedpericyte-like cells (29 transcripts) and primary brain pericytes (26transcripts). A detailed listing of genes and FPKM values can be foundin Table 4.

FIGS. 4A-4F. hPSC-derived pericyte-like cell assembly with endothelialcells. A) Self-assembly schematic. hPSC-derived pericyte-like cellsself-assemble with HUVECs to form vascular cords. B) Confocalimmunocytochemistry images of primary pericytes and H9-derivedpericyte-like cells (NG2) aligning with and extending processes alongHUVEC cords (CD31). Hoechst nuclear counter stain (blue) is alsoincluded. Scale bars: 50 μm. C) Immunocytochemistry images of HUVECsalone or cultured with HEK293 fibroblasts (+HEK293), primary human brainpericytes (+Primary Pericytes), CS03n2-derived pericyte-like cells(+CS03n2), H9-derived pericyte-like cells (+H9), or IMR90C4-derivedpericyte-like cells (+IMR90C4). Hoechst nuclear counter stain (blue) isalso included. Scale bars: 100 μm. D) Representative bright field imagesof HUVECs alone or cultured with the various cell types. Scale bars: 300μm. E) Quantification of the average segment lengths from bright fieldimages in panel D. Plotted are means±SEM of three independentpericyte-like cell differentiations. *P<0.05 vs. HUVEC monoculture;ANOVA followed by Dunnett's test. F) Quantification of the number ofsegments per field normalized to HUVEC monoculture from bright fieldimages in panel D. Plotted are means±SEM of three independentpericyte-like cell differentiations. *P<0.05 vs. HUVEC monoculture;ANOVA followed by Dunnett's test.

FIGS. 5A-5I. Measurement of the effects of hPSC-derived pericyte-likecells on BBB phenotypes. A) Schematic of Transwell setup for co-cultureassays. B) Maximum TEER achieved by IMR90C4-derived BMEC monoculture orco-culture with 3T3 mouse fibroblasts, primary human brain pericytes,H9-derived pericyte-like cells, CS03n2-derived pericyte-like cells, orIMR90C4-derived pericyte-like cells. Plotted are the means±SEM of atleast 3 independent differentiations per condition. *P<0.05 vs.monoculture; ANOVA followed by Dunnett's test. C) Sodium fluoresceinpermeability for IMR90C4-derived BMECs in monoculture or co-culture withcell types as described in B. Plotted are the means±SEM of at least 3independent differentiations per condition, *P<0.05 vs. monoculture;ANOVA followed by Dunnett's test. D) Representative images of occludinimmunocytochemistry of BMECs cultured for 48 h in EC medium (Mono) or ECmedium conditioned by the cell types described in B. Enlarged example ofa frayed junction is inset in the monoculture panel. Scale bar: 25 μm.E) Quantification of occludin area fraction index for the samplesdescribed in D. Plotted are the means±SEM of 3 independentdifferentiations. No significant difference by ANOVA. F) Quantificationof frayed junctions visualized by occludin immunocytochemistry for thesamples described in D. Plotted are the means±SEM of 3 independentdifferentiations. * P<0.05 vs. monoculture; ANOVA followed by Dunnett'stest. G) Accumulation of Alexa-488-tagged 10 kDa dextran inIMR90C4-derived BMECs following 48 hours of co-culture with cell typesas described in B. All results are normalized to BMEC monoculturecontrol. Plotted are the means±SD of 3 Transwells. Results arerepresentative of 3 independent differentiations. * P<0.05 vs.monoculture; ANOVA followed by Dunnett's test. H,I) Transcytosis ofAlexa-488-tagged 10 kDa dextran at 37° C. (H) or 4° C. (I) acrossIMR90C4-derived BMECs following 48 hours co-culture with the cell typesas described in B. All results are normalized to BMEC monoculturecontrol. Plotted are the means±SD from 3 Transwells. Results arerepresentative of 3 independent differentiations. * P<0.05 vs.monoculture; ANOVA followed by Dunnett's test. No significantdifferences at 4° C. by ANOVA.

FIGS. 6A-6C. FIG. 6 : IMR90C4-derived pericyte-like cells integrate intoa complete isogenic NVU model. A) Schematic of IMR90C4-derived BMECco-culture set up with IMR90C4-derived NVU cell types. B) Maximum TEERachieved in IMR90C4-derived BMECs following monoculture or co-culture.Plotted are the means±SD from 3 Transwells. Results are representativeof 3 independent differentiations. * P<0.05 vs. monoculture; #P<0.05 vs.pericyte-like cell co-culture; % P<0.05 vs. astrocyte/neuron co-culture;ANOVA followed by Tukey's HSD test. C) Sodium fluorescein permeabilityin IMR90C4-derived BMECs following 48-hours of monoculture orco-culture. Plotted are the means±SD from 3 Transwells. Results arerepresentative of 3 independent differentiations. *P<0.05 vs.monoculture; ANOVA followed by Tukey's HSD test.

FIGS. 7A-7J: Generation of multipotent NCSC populations from multiplehPSC lines. A) Immunocytochemistry images of small molecule screen (n=1)on HNK1 and p75-NGFR expression in cells differentiated from H9 hESCs.Cells were cultured fifteen days in E6+10 ng/mL FGF2+22.5 μg/mLheparin+10 μM SB431542+CHIR99012±dorsomorphin at the indicatedconcentrations. Hoechst nuclear counter stain (blue) is also included.Scale bars: 100 μm. B) Immunocytochemistry images of IMR90C4 iPSCsdifferentiated in E6-CSFD probed for the presence of HNK1 and p75-NGFRat D15. Hoechst nuclear counter stain (blue) is also included. Scalebars: 100 μm. C) Immunocytochemistry images of CS03n2 iPSCsdifferentiated in E6-CSFD probed for the presence of HNK1 and p75-NGFRat D15. Hoechst nuclear counter stain (blue) is also included. Scalebars: 100 μm. D) AP-2 immunocytochemistry images for IMR90C4-derivedNCSCs at D15. Hoechst nuclear counter stain (blue) is also included.Scale bar: 100 μm. E) AP-2 immunocytochemistry images for CS03n2-derivedNCSCs at D15. Hoechst DNA nuclear stain (blue) is also included. Scalebar: 100 μin. F) Temporal PCR analysis of pluripotency (NANOG, POU5F1)and NCSC (TFAP2A, B3GAT1, NGFR, SOX9, SOX10) transcripts in IMR90C4 andCS03n2 iPSCs and NCSC progeny. G) Flow cytometry analysis ofIMR90C4-derived NCSCs. Panels include NCSC (HNK1⁺/p75-NGFR⁺) purityprior to MACS (panel i), and NCSC purity following MACS (panel ii).Inset percentages are included in each quadrant. Quantitation is shownin Table 1. H) Flow cytometry analysis of CS03n2-derived NCSCs. Panelsinclude NCSC (HNK1⁺/p75-NGFR⁺) purity prior to MACS (panel i), and NCSCpurity following MACS (panel ii). Inset percentages are included in eachquadrant. Quantitation is shown in Table 1. I) Immunocytochemistryanalysis of IMR90C4-derived NCSCs subsequently differentiated inperipheral neuron medium. Resultant cells were positive for βIII-tubulinand peripherin expression. Hoechst nuclear counter stain (blue) is alsoincluded. Scale bar: 100 μm. J) IMR90C4-derived NCSCs could bedifferentiated into mesenchymal derivatives, including Oil Red O stainedadipocytes (panel i, red), Alizarin red stained osteocytes (panel ii,red), and Alcian blue stained chondrocytes (panel ii, blue).

FIGS. 8A-8R: Serum treatment directs iPSC-derived NCSCs towards muralcells. A, B) Representative PDGFRβ and NG2 flow cytometry plots forIMR90C4-derived NCSCs treated for 9 days with E6+10% FBS medium. C, D)Representative PDGFRβ and NG2 flow cytometry plots for CS03n2-derivedNCSCs treated for 9 days with E6+10% FBS medium. Quantitative resultscan be found in FIG. 1J. E) PDGFRβ and NG2 immunocytochemistry ofIMR90C4-derived NCSCs (D16) and mural cells (D22). Hoechst nuclearcounter stain (blue) is also included. Scale bar: 200 μm. F) Calponinand SM22α immunocytochemistry of IMR90C4-derived NCSCs (D16) and muralcells (D22). Hoechst nuclear counter stain (blue) is also included.Scale bar: 200 μm. G) α-SMA immunocytochemistry of IMR90C4-derived NCSCs(D16) and mural cells (D22). Hoechst nuclear counter stain (blue) isalso included. Scale bars: 200 μm. H,I) CD13 and desminimmunocytochemistry of IMR90C4-derived mural cells (D22). Hoechstnuclear counter stain (blue) is also included. Scale bars: 200 μm. J)PDGFRβ and NG2 immunocytochemistry of CS03n2-derived mural cells (D22).Hoechst nuclear counter stain (blue) is also included. Scale bar: 200μm. K) Calponin and SM22α immunocytochemistry of CS03n2-derived muralcells (D22). Hoechst nuclear counter stain (blue) is also included.Scale bar: 200 μm. L) α-SMA immunocytochemistry of CS03n2-derived muralcells (D22). Hoechst DNA nuclear stain (blue) is also included. Scalebars: 200 μm. M,N) CD13 and desmin immunocytochemistry of CS03n2-derivedpericyte-like cells (D22). Hoechst nuclear counter stain (blue) is alsoincluded. Scale bars: 200 μm. O,P) CD13 and desmin immunocytochemistryof H9-derived NCSCs (D16). Hoechst nuclear counter stain (blue) is alsoincluded. Scale bars: 200 μm. Q,R) CD13 and desmin immunocytochemistryof primary brain pericytes. Hoechst nuclear counter stain (blue) is alsoincluded. Scale bars: 200 μm.

FIGS. 9A-9F: Supplemental analysis of hPSC-derived pericyte-like cells.A,B) Analysis of cells obtained by culturing NCSCs in E6,E6+TGFβ1+PDGF-BB, or E6+10% FBS for 6 days. Calponin, SM22α, and α-SMAimmunocytochemistry. Hoechst nuclear counter stain (blue) is alsoincluded. Scale bars: 200 μm. C,D,E) Long-term maintenance ofhPSC-derived pericyte-like cells. PDGFRβ, NG2, calponin, SM22α, andα-SMA immunocytochemistry of H9-derived pericyte-like cells maintainedin E6+10% FBS to D45. Hoechst nuclear counter stain (blue) is alsoincluded. Scale bars: 200 μm. F) Temporal PCR analysis of mural andpericyte transcripts for the differentiating IMR90C4 and CS03n2 iPSClines.

FIGS. 10A-10D. Supplemental analysis of BMEC/hPSC-derived pericyte-likecell co-cultures. A) PDGFRβ and NG2 immunocytochemistry of hPSC-derivedpericyte-like cells following 48 hours of co-culture with iPSC-derivedBMECs. Hoechst nuclear counter stain (blue) is also included. Images arerepresentative of two independent differentiations. Scale bars: 100 μm.B) Western blot analysis of occludin and claudin-5 expression iniPSC-derived BMECs cultured alone or co-cultured with primary brainpericytes, IMR90C4-derived pericyte-like cells, or 3T3s. Quantificationof occludin and claudin-5 expression after normalized to β-actin signaland to monoculture expression levels. Plotted are the means±SD from 3Transwells from a single differentiation. No significant differences byANOVA. C) Representative images of claudin-5 immunocytochemistry ofBMECs cultured for 48 h in EC medium (Mono) or EC medium conditioned byprimary brain pericytes or IMR90C4-derived pericyte-like cells. Scalebar: 25 μm. Quantification of claudin-5 area fraction index and frayedjunctions. Plotted are the means±SEM of 3 independent differentiations.No significant difference by ANOVA. D) Confocal microscopy ofmonocultured iPSC-derived BMECs incubated with Alexa 488-tagged 10 kDadextran (green) with EC medium (Mono) or conditioned medium from primarybrain pericytes, IMR90C4-derived pericyte-like cells, or 3T3s. Totaldextran is depicted in green. Surface dextran was labeled with Alexa 647(red), with little observed signal. Thus, the observed green signal is aresult of internalized dextran. Hoechst nuclear counter stain (blue) isalso included. Scale bar: 10 μm.

FIGS. 11A-11C. Measurement of the effects of hPSC-derived pericyte-likecells on primary rat BMEC phenotypes. A) TEER profile of primary ratBMECs either in monoculture or co-culture with primary brain pericytes,IMR90C4-derived pericyte-like cells, or 3T3s. Plotted are means±SD ofthree Transwells from a single rat BMEC isolation. *P<0.05IMR90C4-derived pericyte-like cell co-culture vs. monoculture; #P<0.05primary pericyte co-culture vs. monoculture; ANOVA followed by Dunnett'stest. B,C) Accumulation (B) or transcytosis (C) of Alexa 488-tagged 10kDa dextran in primary rat BMECs following co-culture with cell types asdescribed in A. All results normalized to BMEC monoculture control.Plotted are the means±SD of 3 Transwells from a single rat BMECisolation. * P<0.05 vs. monoculture; ANOVA followed by Dunnett's test.

FIG. 12A-12D. NCSCs maintained in E6-CSFD retain neural crest markerexpression and do not develop pericyte marker expression. A) Expression(FPKM) of selected transcripts in D15 NCSCs, D55 NCSCs (maintained inE6-CSFD for an additional 40 days), and all D25 pericyte-like cellsamples (“H”). B) p75-NGFR immunocytochemistry analysis of NCSCsmaintained in E6-CSFD for 3 months. Hoechst nuclear counter stain (blue)is also included. Scale bar: 200 μm. C) NG2 immunocytochemistry analysisof NCSCs maintained in E6-CSED for 3 months. Hoechst nuclear counterstain (blue) is also included. Scale bar: 200 μm. D) NG2immunocytochemistry analysis of H9-derived D22 pericyte-like cells,processed alongside and identically to the NCSC sample above. Hoechstnuclear counter stain (blue) is also included. Scale bar: 200 μm.

FIGS. 13A-13E: hPSC-derived pericyte-like cell assembly with brainendothelial cells. A) Self-assembly schematic. hPSC-derivedpericyte-like cells self-assemble with hBMECs to form vascular cords. B)Confocal immunocytochemistry images of primary pericytes and H9-derivedpericyte-like cells (NG2) aligning with and extending processes alonghBMEC cords (CD31). Hoechst nuclear counter stain (blue) is alsoincluded. Scale bars: 50 μm. C) Immunocytochemistry images of hBMECsalone or cultured with HEK293 fibroblasts (+HEK293), primary human brainpericytes (+Primary Pericytes), CS03n2-derived pericyte-like cells(+CS03n2), H9-derived pericyte-like cells (+H9), or IMR90C4-derivedpericyte-like cells (+IMR90C4). Hoechst nuclear counter stain (blue) isalso included. Scale bars: 200 μm. D) Representative bright field imagesof HUVECs alone or cultured with the various cell types. Scale bars: 200μm. E) Quantification of the average segment lengths from bright fieldimages in panel D. Plotted are means±SD of three imaging fields from onewell. * P<0.05 vs. hBMEC monoculture; ANOVA followed by Dunnett's test.F) Quantification of the number of segments per field normalized tohBMEC monoculture from bright field images in panel D. Plotted aremeans±SD of three imaging fields from one well. * P<0.05 vs. HUVECmonoculture; ANOVA followed by Dunnett's test.

DETAILED DESCRIPTION OF THE INVENTION

Abbreviations: blood-brain barrier, BBB; brain microvascular endothelialcells, BMECs; central nervous system, CNS; E6 medium supplemented withCHIR99021, SB431542, FGF2, and dorsomorphin, E6-CSFD; endothelial growthfactor medium 2, EGM-2; human embryonic stem cells, hESCs; humanpluripotent stem cells, hPSCs; induced pluripotent stem cells, iPSCs;neural crest stem cells, NCSC; neurovascular unit, NVU; vascular smoothmuscle cells, vSMCs

In General

As discussed above, brain pericytes play an important role in theformation and maintenance of the neurovascular unit (NVU), and theirdysfunction has been implicated in central nervous system (CNS)disorders. While human pluripotent stem cells (hPSCs) have been used tomodel other components of the NVU including brain microvascularendothelial cells (BMECs), astrocytes, and neurons, cells having brainpericyte-like phenotypes have not been described.

In the work supporting the present application, we generated neuralcrest stem cells (NCSCs), the embryonic precursor to forebrainpericytes, from human pluripotent stem cells (hPSCs) and subsequentlydifferentiated NCSCs into brain pericyte-like cells. The brainpericyte-like cells expressed marker profiles that closely resembledprimary human brain pericytes but lack the ACTA2 marker, which is foundin primary pericytes. As disclosed below in the Examples, the brainpericyte-like cells self-assembled with endothelial cells to supportvascular tube formation. Importantly, the brain pericyte-like cells alsoinduced blood-brain barrier (BBB) properties in BMECs by at least 20%,preferably at least 30% or 50%, including barrier enhancement andreduction of transcytosis. Finally, brain pericyte-like cells wereincorporated with iPSC-derived BMECs, astrocytes, and neurons to form anisogenic human NVU model that should prove useful for the study of theBBB in CNS health, disease, and therapy.

U.S. Ser. No. 13/793,466 (Publication U.S.2017/025935), Ser. No.13/218,123 (U.S. Pat. No. 8,293,495) and Ser. No. 16/092,450(Publication U.S.2019/0093084) are drawn to related technology andshould be incorporated by reference herein. U.S. Ser. No. 13/218,123discloses a preferred method of creating an isogenic BBB model (i.e.,all of the cell types present are derived for a single patient iPSCline), which comprises BMECs, neurons, and astrocytes. At the time Ser.No. 13/218,123 was filed, the inventors had not finalized their pericytedifferentiation protocol, nor had they shown that the addition ofpericytes to the BBB resulted in a functional improvement. U.S. Ser. No.13/793,466 discloses an improved BBB model that incorporates retinoicacid (RA). Both of these disclosures provide context for the use of thepericyte-like cells of the present invention.

The present invention also provides methods for obtaining a brainpericyte-like cell population and populations of the cells, includingprogenitor cells. The disclosure also include isogenic BBB models.

Cell Populations

In one embodiment, the present invention is a population of brainpericyte-like cells, wherein the cells expresses pericyte markers but donot express ACTA2 and wherein the cells are generated from hPSC.

By “pericyte markers,” we mean the markers listed in the Examples below,including FIG. 2 (e.g., 2E and 2B). Note that the brain pericyte-likecells of the present invention do not express detectable ACTA2 (see FIG.2B). Exemplary pericyte markers include CNN1, NG2, and PDGFRB. Forexample, the brain pericyte-like cells of the present invention areNG2⁺PDGFRB⁺ACTA2⁻ cells. Exemplary pericyte markers that are expressedon the brain pericyte-like cells of the invention demonstrated in FIG.2E and FIG. 9A include expression of one or more transcripts of pericytemarkers selected from the group consisting of CSPG4, PDGFRB, CNN1,TAGLN, ANPEP, TBX18, ABCC9 and KCNJ8. In one suitable example, the brainpericyte-like cells express the transcripts of at least ABCC9 and KCNJ8.

Any appropriate method can be used to detect expression of biologicalmarkers characteristic of cell types described herein. For example, thepresence or absence of one or more biological markers can be detectedusing, for example, RNA sequencing, immunohistochemistry, polymerasechain reaction, qRT-PCR, or other technique that detects or measuresgene expression. Suitable methods for evaluating the above-markers arewell known in the art and include, e.g., qRT-PCR, RNA-sequencing, andthe like for evaluating gene expression at the RNA level. Quantitativemethods for evaluating expression of markers at the protein level incell populations are also known in the art. For example, flow cytometryis typically used to determine the fraction of cells in a given cellpopulation that express (or do not express) a protein marker of interest(e.g., NG2, PDGFRB). In some cases, cell populations obtained by thedifferentiation methods of this disclosure comprise at least 80%, 85%,90%, 95% and preferably at least 98% NG2+PDGFRB+ACTA2-brainpericyte-like cells.

The Examples below describe suitable hPSC lines. The human pluripotentstem cells may be embryonic stem cells or induced pluripotent stem cells(iPSCs). The present invention is also meant to employ iPSC lines arethat developed from individual patients or disease models.

As used herein, “pluripotent stem cells” appropriate for use accordingto a method of the invention are cells having the capacity todifferentiate into cells of all three germ layers. Suitable pluripotentcells for use herein include human embryonic stem cells (hESCs) andhuman induced pluripotent stem cells (iPSCs). As used herein, “embryonicstem cells” or “ESCs” mean a pluripotent cell or population ofpluripotent cells derived from an inner cell mass of a blastocyst. SeeThomson et al., Science 282:1145-1147 (1998). These cells express Oct-4,SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81, and appear as compact colonieshaving a high nucleus to cytoplasm ratio and prominent nucleolus. ESCsare commercially available from sources such as WiCell ResearchInstitute (Madison, Wis.). As used herein, “induced pluripotent stemcells” or “iPSCs” mean a pluripotent cell or population of pluripotentcells that may vary with respect to their differentiated somatic cell oforigin, that may vary with respect to a specific set ofpotency-determining factors and that may vary with respect to cultureconditions used to isolate them, but nonetheless are substantiallygenetically identical to their respective differentiated somatic cell oforigin and display characteristics similar to higher potency cells, suchas ESCs. See, e.g., Yu et al., Science 318:1917-1920 (2007),incorporated by reference in its entirety. Induced pluripotent stemcells exhibit morphological properties (e.g., round shape, largenucleoli and scant cytoplasm) and growth properties (e.g., doubling timeof about seventeen to eighteen hours) akin to ESCs. In addition, iPSCsexpress pluripotent cell-specific markers (e.g., Oct-4, SSEA-3, SSEA-4,Tra-1-60 or Tra-1-81, but not SSEA-1). Induced pluripotent stem cells,however, are not immediately derived from embryos. As used herein, “notimmediately derived from embryos” means that the starting cell type forproducing iPSCs is a non-pluripotent cell, such as a multipotent cell orterminally differentiated cell, such as somatic cells obtained from apost-natal individual.

The present invention provides an in vitro population of brainpericyte-like cells derived from human pluripotent stem cells, whereinthe brain pericyte-like express pericyte markers but do not expressACTA2. In one embodiment, the pericyte markers include one or more ofthe following markers CNN1, NG2, and PDGFRB. In a preferred example, thein vitro derived population is NG2⁺PDGFRB⁺ brain pericyte-like cells.Alternatively, the in vitro derived population is NG2⁺PDGFRB⁺ACTA2⁻population of cells. The methods described below for generating thebrain pericyte-like cells provide a substantially pure population ofcells, for example, the methods provides a population that is at least90% NG2⁺PDGFRB⁺.

As described more in the examples below, the brain pericyte-like cellsfurther express calponin and SM22α but do not express α-SMA,distinguishing the cells from smooth muscle and other cell types. ThehPSC-derived brain pericyte-like cells generated by the methodsdescribed herein express one or more transcripts of pericyte markersselected from the group consisting of CSPG4, PDGFRB, CNN1, TAGLN, ANPEP,TBX18, ABCC9 and KCNJ8. These hPSC-derived brain pericyte-like cells arecapable of inducing pericyte-driven phenomena in BMEC, including theenhancement of barrier properties (e.g., increased TEER for the BMECs)and reduction of transcytosis (e.g., reduction in the ability totransport molecules across the in vitro made BMEC barrier).

The brain pericyte-like cells of the present invention typically arecapable of associating with vascular networks. In the Example and asdemonstrated in FIG. 4 , the brain pericyte-like cells can self-assemblewith HUVECs to form vascular cords and a method of measuring thisphenomenon. This is an important component of pericyte function. In oneembodiment, the present disclosure provides in vitro vascular tubescomprising NG2⁺PDGFRB⁺ACTA2⁻ brain pericyte-like cells derived from PSCsand human umbilical vein endothelial cells (HUVECs) or immortalizedhuman BMECs (hBMECs).

The present invention provides methods of in vitro producing vascularcords for study. The hPSC-derived brain pericyte-like cells are platedon coated plates (e.g., MATRIGEL™, vitronectin, a vitronectin fragment,or a vitronectin peptide, or Synthemax® coated plates, preferablyMATRIGEL™) with human umbilical vein endothelial cells (HUVECs, e.g.,CD31+HUVECs) or immortalized human BMECs (hBMECs) self-associated withHUVECs and hBMECs much like primary human brain pericytes (FIG. 4B-C;FIG. 13B-C). After 24 hours in co-culture, hPSC-derived pericyte-likecells exhibited high NG2 expression and aligned along the CD31⁺endothelial cell cord perimeter and developed pericyte-like morphologywith stellate-shaped bodies and extended cell processes (FIG. 4B-C). ThehPSC-derived brain pericyte-like cells cocultured with which HUVECs orhBMECs yielded fewer yet longer and more developed cord networks (FIG.4C-F, 13C-F) when compared to HUVECs or hBMECs alone and HUVECs orhBMECs in co-culture with control HEK293 cells (which yielded many smallbranching cords). Culture conditions include plating the hPSC-derivedbrain pericyte-like cells at a ratio of 3:1 (suitable ratios range from4:1 to 2.5:1), in EGM-2 medium for at least 24 hours.

Additionally, the brain pericyte-like cells of the present inventiontypically are capable of inducing pericyte-driven phenomenon in BMEC.Most importantly, this would include the enhancement of barrierproperties and reduction of transcytosis. For example, FIG. 5 shows themeasurement of the effects of the brain pericyte-like cells of thepresent invention on BBB phenotypes. Briefly, hPSC-derived brainpericyte-like cells are co-cultured in a transwell system (e.g.,polystyrene transwell filters with a 0.4 μm pore size) with iPSC-derivedBMECs in endothelial cell (EC) medium without FGF2. The transwellcoculture system is depicted in FIG. 5A, showing an upper compartmentseparated by a transwell insert containing a microporous semi-permeablemembrane that separates the cells in the upper compartment (e.g.,CD31+iPSC-derived BMECs) from the cells in the lower compartment (e.g.,the NG2+PDGFRB+hPSC-derived brain pericyte-like cells of the presentinvention). Suitable EC medium are known in the art and commerciallyavailable (e.g., available from Promocell, R&D Systems, Sigma-Aldrich,ScienCell Research Laboratories, Lonza, among others),

In one embodiment, the present invention is a population of NC SC cellsprepared by the method described below.

Methods

In another embodiment, the present invention is a method of creating apopulation of brain pericyte-like cells, wherein the cells expresspericyte markers but do not express ACTA2 and wherein the cells aregenerated from hPSC. In general, the method comprises the stepsdescribed below in the Examples. Typically, the method begins withculturing or maintaining hPSC in E8 medium. In some embodiments, thisculture step is on coated plates, for example, Matrigel™ coated plates.The Examples below contain a description of E8 medium and a typicalmethod of media preparation. The Examples below disclose a preferredmethod of singularizing the cells using Accutase and preferred seedingdensities. As used herein, the terms “E8 culture medium” and “E8” areused interchangeably and refer to a chemically defined culture mediumcomprising or consisting essentially of DF3S supplemented by theaddition of insulin (20 μg/mL), transferrin (10.67 ng/mL), human FGF2(100 ng/mL), and human TGFβ1 (Transforming Growth Factor Beta 1) (1.75ng/mL). The medium can be prepared based on the formula in previouspublication (Chen et al., (2011) Nature Methods. 8(4), 424-429). As analternative, the medium is also available from ThermoFisher/LifeTechnologies Inc. as Essential 8, or from Stem Cell Technologies asTeSR-E8.

Further differentiation steps include using E6 medium that is describedherein and in U.S. Patent Publication No. 2014/0134732. Preferably, thechemically defined medium comprises DMEM/F-12. E6 medium containsDMEM/F12; L-ascorbic acid-2-phosphate magnesium (64 mg/l); sodiumselenium (14 μg/l); insulin (20 mg/l); NaHCO₃ (543 mg/l); andtransferrin (10.7 mg/l). E6-CSFD is E6 medium supplemented withCHIR99021, SB431542, FGF2, and dorsomorphin. Suitable ranges of thefactors are for inclusion in E6 medium to produce E6-CSFD mediaincludes, for example, about 0.5-5 μM CHIR99021 (preferably 1 μM), aGSK3β inhibitor to promote WNT signaling; 5-20 μM SB431543 (preferably10 μM), an ALKS antagonist to inhibit Activin/Nodal/TGFβ signaling;5-100 ng/ml FGF2 (preferably 10 ng/mL); and about 0.5-2 μM dorsomorphin(preferable 1 μM), a BMP type I receptor inhibitor. One exemplaryformulation of E6-CSFD is 1 μM CHIR99021; 10 μM SB431543; 10 ng/mL FGF2(E6-CSF); and 1 μM CHIR99021, and 1 μM dorsomorphin.

One would then culture the in vitro hPSCs described above in E6-CSFDmedium for about 15 days to produce a population comprising NCSCs.Suitable, the cells can be cultured for at least 15 days, and can bemaintained in culture to about 60 days.

One would then typically sort and re-plate the NCSC cells, which expressHNK1 and p75-NGFR (e.g., p75-NGFR⁺HNK1⁺NCSCs) and can be differentiatedto various mesenchymal derivatives such as osteocytes, adipocytes andchondrocytes under different culturing conditions as described in theExamples below.

One would then culture the NCSC cells (e.g., p75-NGFR⁺HNK1⁺ NCSCs) in E6medium with an addition of serum (preferably 1-10%) for about 11 days,wherein a population of PSC-derived brain pericyte-like cells thatexpress pericyte markers but do not express ACTA2 can be isolated, thiscell population is described in more detail above. These could befurther cultured as brain pericyte-like cells for about 30 additionaldays (e.g., from 11 to 41 days).

In one embodiment, a method of creating a population of p75-NGFR⁺HNK⁺NSCs from human pluripotent stem cells is provided. The method comprisesculturing hPSC in E6-CSFD medium for about 15 days to producedp75-NGFR⁺HNK⁺ NCSC cells, subsequently sorting p75-NGFR⁺ cells from thepopulation and re-plating the p75-NGFR⁺ cells of step to produce apopulation of p75-NGFR⁺NCSCs. Methods of sorting the p75-NGFR⁺ cells areknown to one skilled in the art and include, but are not limited to,fluorescence activated cell sorting (FACS) and magnetic-activated cellsorting (MACS), among others. A preferred method of sorting the cells isMACS.

The population of cells produced from PSCs is a p75-NGFR⁺HNK⁺AP-2⁺NCSCswhich are able to be maintained in culture, e.g., the cells are able todouble at least 5 times in culture and still maintain expressionp75-NGFR⁺, HNK⁺, and AP-2⁺ within the cells. These NCSCs are able to bemaintained in culture for at least five passages and maintainNGFR⁺HNK⁺AP-2⁺ marker expression and do not express pericyte markers(e.g., NG2⁻, PDBGFR⁻, etc). These NCSCs produced by the method describedherein are able to maintain the potential to differentiate into neuronsand mesenchymal cells, as demonstrated in the Examples below.

In vitro derived hPSC-derived brain pericyte-like cells can be producedfrom these NCSC cells. The method of producing hPSC-derived brainpericyte-like cells (e.g., NG2+PDGFRB+hPSC-derived brain pericyte-likecells) comprises culturing the NCSC cells (e.g.,p75-NGFR⁺HNK⁺AP-2⁺NCSCs) in E6 media with an addition of 1-10% serum forabout 11 days, wherein the NCSCs produce a population of brainpericyte-like cells that express NG2, and PDGFRB but do not expressACTA2.

In another embodiment, the disclosure provides a method of creating apopulation of hPSC-derived brain pericyte-like cells comprising thesteps of (a) culturing hPSC in E6-CSFD medium for about 15 days toproduced p75-NGFR⁺HNK⁺ NCSC cells, (b) sorting p75-NGFR⁺ cells andre-plating the p75-NGFR⁺ cells to produce a population ofp75-NGFR⁺NCSCs, and (c) culturing the cells of step (b) in E6 media withan addition of 1-10% serum for about 11 days to generate a population ofbrain pericyte-like cells that express pericyte markers but do notexpress ACTA2 is produced. The population of hPSC-derived brainpericyte-like cells express one or more marker selected from the groupconsisting of CNN1, NG2, and PDGFRB, e.g., NG2⁺PDGFRB⁺hPSC-derived brainpericyte-like cells. The population of brain pericyte-like cells can befurther characterized by the express one or more transcripts of pericytemarkers selected from the group consisting of CSPG4, PDGFRB, CNN1,TAGLN, ANPEP, TBX18, ABCC9 and KCNJ8. This method produces anhPSC-derived brain pericyte-like cell population that is at least 90%NG2⁺PDGFRB⁺.

As discussed above, these hPSC-derived brain pericyte-like cells can beused in a variety of assays and models, including an isogenic human NVUmodel and an isogenic BBB model.

An Isogenic BBB Model

In another embodiment, the present invention is a BBB model created bythe method disclosed in U.S. Ser. No. 13/793,466 (U.S.2017/025935) orany other method of using pericytes in the creation of a BBB model(multicellular BBB model). Most preferably, the model is an isogenicmodel. The term “isogenic” as used herein, refers to cells originatingor differentiated from the same subject or same line of humanpluripotent stem cells (hPSCs). The cells are not exposed to cells of analternate genetic origin as the model is being prepared. In the presentinvention, hPSC derived brain pericyte-like cells are co-cultured withBMECs derived from the same hPSC source to create an isogenic bloodbrain barrier model.

In a previous U.S. patent application (Ser. No. 13/155,435, U.S. PatentPublication No. 2012/0015395, incorporated herein by reference),Applicants demonstrated that human pluripotent stem cells could bedifferentiated into brain microvascular endothelial cells (BMECs). Inanother previous U.S. patent application (Ser. No. 13/793,466, U.S.Patent Publication No. 2014/0127800, incorporated herein by reference).

The BBB model contemplated herein would be entirely derived from invitro hPSC-derived cells. The hPSC-derived brain pericyte-like cellsdescribed herein can be used in the BBB model using a transwell systemto coculture BMECS and supporting cells (e.g., pericytes,) mimic a bloodbrain barrier using in vitro iPSC-derived BMECs (CD31⁺BMECs). This modelis described in US that provide Ser. No. 13/793,466. BBB models can beused to help elucidate the role of the BBB in brain development,function, and disease, and to develop potential therapeutic approaches.However, BBB models using primary and transformed BMECs tend tode-differentiate and lose their barrier properties once they are removedfrom the brain microenvironment and often exhibit sub-par BBB phenotypes(Weksler et al. 2005, Förster et al. 2008, Man et al. 2008, Calabria &Shusta 2008). The brain pericyte-cells of the present invention wouldallow for the use of a fully in vitro derived BBB from hPSCs.

EXAMPLES Directed Differentiation of hPSCs to NCSCs in Low ProteinMedium

We first assessed the capability of E6, a reduced factor medium, tosupport differentiation of H9 human embryonic stem cells (hESCs) andIMR90C4 and CS03n2 induced pluripotent stem cells (iPSCs) to NCSCs. H9hESCs were cultured for 15 days in E6 medium supplemented with heparinand pathway modulators previously implicated in hPSC differentiation toNCSCs (48): 1 μM CHIR99021, a GSK3β inhibitor to promote WNT signaling;10 μM SB431543, an ALK5 antagonist to inhibit Activin/Nodal/TGFβsignaling; and 10 ng/mL FGF2 (E6-CSF). However, E6-CSF failed to producep75-NGFR⁺/HNK1⁺ NCSCs, and increasing CHIR99021 concentration (2 μM) didnot aid in inducing p75-NGFR expression (FIG. 7A).

BMP signaling during hPSC differentiation to NCSC can inhibit NCSCformation, and WNT signaling activation can induce downstream BMPsignaling in hPSCs (46); however, the requirement of BMP inhibition inNCSC differentiation has been variable (42, 46). To examine the effectsof BMP inhibition on hPSC differentiation to NCSCs in minimal medium,E6-CSF medium was supplemented with 1 μM dorsomorphin, a BMP type Ireceptor inhibitor, to generate E6-CSFD. With BMP inhibition, H9 hESCsprogressed to p75-NGFR⁺/HNK1⁺ NCSCs that also expressed AP-2 after 15days of E6-CSFD treatment (FIG. 1A-C,J; FIG. 7D,E). E6-CSFD also droveNCSC formation in IMR90C4 and CS03n2 iPSC lines (FIG. 1J; FIG. 7B-E). H9and CS03n2 hPSCs yielded cultures comprising ˜90% NCSCs, while purity ofIMR90C4-derived NCSCs was frequently lower (FIG. 1J). Temporal mRNAanalysis confirmed loss of pluripotency by D15 of E6-CSFD treatment, asindicated by loss of NANOG and POU5F1 pluripotency transcripts (FIG. 1D;FIG. 7F). In addition, after 15 days of E6-CSFD treatment, thedifferentiation mixture expressed NCSC-associated transcripts, includingTFAP2A, SOX9, SOX10, B3GAT1 (HNK1) and NGFR (FIG. 1D; FIG. 7F). At D15of E6-CSFD treatment, cells had undergone approximately 7 populationdoublings (FIG. 1E), corresponding to over 100 NCSCs per input hPSC.

To purify NCSCs from the differentiation cultures, day 15 NCSCs werepositively selected using anti-p75-NGFR magnetic activated cell sorting(MACS). MACS enriched p75-NGFR⁺/HNK1⁺ NCSC populations above 95% for allthree hPSC lines tested (FIG. 1F,J; FIG. 7G,H). Sorted NCSCs retainedp75-NGFR and HNK1 expression following replating (FIG. 1G). In addition,treating NCSCs with N2 medium supplemented with BDNF, GDNF, NT-3, andNGF-β yielded βIII-tubulin⁺/peripherin⁺ peripheral neurons (FIG. 1H;FIG. 7I). We additionally expanded sorted NCSCs for 11 days and thendifferentiated these cells to mesenchymal derivatives: Oil Red O⁺adipocytes were obtained by treating NCSCs with insulin, IBMX, anddexamethasone, Alcian blue⁺ chondrocytes using pellet culture andTGFβ1-containing chondrogenic medium, and Alizarin red⁺ osteocytes usingdexamethasone, glycerophosphate, and ascorbic acid (FIG. 1I; FIG. 7J).Taken together, these data demonstrate that reduced factor, low proteinE6-CSFD medium directs hPSCs to NCSCs over a 15-day differentiationperiod, and that MACS-purified NCSCs retain the potential to form NCSCderivatives.

Serum Treatment Directs hPSC-derived NCSCs to Mural Cell Lineages

We subsequently identified differentiation conditions capable of drivingNCSCs to mural cell lineages (FIG. 2A), as defined by coexpression ofPDGFRβ and NG2 (40, 49). PDGFRβ was expressed in D15 NCSCs (FIG. 2C) andin replated cells one day following MACS (D16), but NG2 expression wasabsent in both of these cell populations (FIG. 2C,F). Given theimportance of platelet-derived growth factor-BB (PDGF-BB) and TGFβ1 inmural cell development (50, 51), we first tested if these factors couldinduce NG2 expression in NCSCs while also maintaining PDGFRβ expression.Culture of NCSCs for six days in E6 medium generated cells that werePDGFRβ positive but NG2 expression was not observed (FIG. 2B).Supplementation of E6 medium with PDGF-BB and TGFβ1 did not induce NG2expression. However, when E6 medium was supplemented with 10% FBS,resultant cells expressed both PDGFRβ and NG2 (FIG. 2B). Comparingdifferentiation in E6+10% FBS on uncoated tissue culture polystyrene(TCPS) to gelatin-coated TCPS, which has previously been reported asconducive to mural cell differentiation (52), the uncoated substrateyielded a qualitatively larger fraction of cells that expressed PDGFRβand NG2 (FIG. 2B). Given the capacity for E6+10% FBS on uncoatedsubstrate to direct hPSC-derived NCSCs to PDGFRβ⁺/NG2⁺ mural cells, wefurther evaluated these cells.

The temporal evolution of hPSC-derived NCSCs to PDGFRβ⁺/NG2⁺ mural cellsusing E6+10% FBS was examined over a 9 day period (D16-D25). At D15 ofdifferentiation, 92.4±1.1% of H9-derived NCSCs expressed PDGFRβ, andafter 9 days of serum treatment, nearly all cells were PDGFRβ⁺(99.6±0.2%) (FIG. 2C-D), with expression of PDGFRB transcript present inD15 NCSCs and throughout the differentiation in serum (FIG. 2E). Incontrast, despite the fact that the NG2-encoding CSPG4 transcript wasexpressed in D15 NCSCs (FIG. 2E), NG2 protein was not detected at thistime point by flow cytometry (FIG. 2C). However, the percentage of cellsexpressing NG2 increased over the 9 day differentiation period, withnearly all cells becoming NG2⁺ (99.4±0.3% at D25, P<0.05 vs. D15) (FIG.2C-D). The E6+10% FBS differentiation scheme also generated at least˜90% PDGFRβ⁺ and NG2⁺ cells in IMR90C4- and CS03n2-derived NCSCsfollowing nine days of E6+10% FBS treatment (D25; FIG. 1J; FIG. 8A-D).At D22, this procedure yielded a roughly ten-fold expansion in muralcells (9.5±1.3 mural cells per sorted NCSC for six independentdifferentiations).

To further probe the transition of hPSC-derived NCSCs to pericyte-likecells, we examined the temporal evolution of transcripts that have beenassociated with pericytes and other mural cells. H9 hESCs expressed CNN1(calponin) and TAGLN (SM22α), which encode contractile proteinsimplicated in early mural cell differentiation (41), as did NCSCs andmural cells (FIG. 2E). At D16, replated hPSC-derived NCSCs expressedSM22α but calponin expression was not observed (FIG. 2G; FIG. 8F,K). ByD22, differentiating hPSC-derived NCSCs exhibited calponin/SM22αcoexpression with cellular localization to contractile fibers (FIG. 2G;FIG. 8F,K). Interestingly, smooth muscle actin (α-SMA) was not detectedin D22 cells treated with E6+10% FBS, although serum transientlyincreased abundance of the transcript (ACTA2) before downregulation(FIG. 2E,H; FIG. 8G,L; FIG. 9F). In contrast, NCSCs treated with E6alone or E6 plus PDGF-BB and TGFβ1 expressed α-SMA in addition tocalponin and SM22α (FIG. 9A,B). In addition, these cells exhibited amorphology similar to smooth muscle cells, with large cell bodies anddistinct cell borders, whereas the cells differentiated in E6+10% FBSwere smaller with numerous projections reminiscent of cultured primarybrain pericytes (FIG. 2G,H). After extended culture in E6+10% FBS (D45),the resultant cells continued to be PDGFRβ⁺/NG2⁺ and expressed calponinand SM22α while α-SMA was still absent (FIG. 9C-E). Primary human brainpericytes expressed all three contractile proteins, and had a morphologysimilar to the serum treated NCSC-derived mural cells (FIG. 2F-H). CD13and desmin were expressed both in D16 NCSCs and D22 mural cells, whileprimary brain pericytes expressed desmin but CD13 expression was weak(FIG. 2I,J; FIG. 8H,I,M-R).

Additional transcript analysis was used to further characterize thedifferentiation process. The mural cell marker, ANPEP (CD13), wasexpressed throughout the differentiation process. While PDGFRB, CSPG4(NG2), CNN1, TAGLN, ANPEP, and TBX18 are mural cell markers expressedthroughout the body, FOXF2 and ZIC1 have been suggested as beingselectively expressed in brain mural cells (53-55). Accordingly, giventhe NCSC origin of the mural cells, FOXF2 and ZIC1 were induced duringthe differentiation (FIG. 2E; FIG. 9F). Until recently, it has beendifficult to use markers to distinguish pericytes from smooth musclecells in brain; however, it has been suggested that ABCC9 and KCNJ8 aretwo transcripts having selective expression in brain pericytes ascompared to smooth muscle (40, 49). ABCC9 levels were biphasic withstrong expression in D15 NCSC and then a re-induction in D25 muralcells. KCNJ8 was expressed fairly uniformly throughout thedifferentiation process (FIG. 2E). Similar results were observed formural cells derived from IMR90C4- and CS03n2-derived NCSCs, although theIMR90C4 mural cells had weaker ZIC1 and ABCC9 signatures (FIG. 9F).Overall, the transcript profile of mural and pericyte-associated genesin the NCSC-derived mural cells was qualitatively very similar to thatof primary human brain pericytes (FIG. 2E).

We next used RNA-sequencing (RNA-seq) to quantify global gene expressionin NCSC-derived mural cells and to evaluate the temporal emergence of apericyte-like population. As expected, unbiased hierarchical clusteringbased on expression (fragments per kilobase of transcript per millionmapped reads, FPKM) of all transcripts revealed the highest similaritybetween NCSC-derived mural cells generated from three independentdifferentiations from H9 hESCs as well as the two differentiations fromIMR90C4 and CS03n2 iPSCs (FIG. 3A, D25 sample cluster). The Pearsoncorrelation coefficients comparing transcript expression in H9-derivedmural cells at D25 to the two replicate H9 differentiations were 0.99and 0.98 (D25 H9-A versus D25 H9-B or H9-C, P<0.0001). Moreover, thePearson correlation coefficients comparing the mural cells derived fromthe H9 hESC line to those derived from IMR90C4 and CS03n2 iPSCs wereboth 0.97 (D25 H9-A versus D25 IMR90 or CS03, P<0.0001). Collectively,these data indicate a highly reproducible differentiation procedureamongst replicated differentiations and hPSC lines. Furthermore,NCSC-derived mural cells at D25 clustered more closely with primarybrain pericytes than with D15 NCSCs, D55 NCSCs that had been maintainedin E6-CSFD following MACS, or hPSCs (FIG. 3A). The Pearson correlationcoefficient between the average transcript expression of all D25NCSC-derived mural cell samples and the average of the primary pericytesamples was 0.89 (P<0.0001), suggesting strong positive associationbetween NCSC-derived mural cells and primary human pericytes. Consistentwith RT-PCR experiments (FIG. 1D; FIG. 2E), temporal analysis oftranscript expression demonstrated downregulation of pluripotencymarkers NANOG and POU5F1, and transient upregulation of NGFR, B3GAT1,SOX9, and SOX10, as well as the cranial neural crest marker ETS1, in D15NCSCs (FIG. 3B). We also observed gradual induction of CSPG4, PDGFRB,CNN1, TAGLN, ANPEP, TBX18, ABCC9, and KCNJ8 over the time course ofE6+10% FBS treatment, and transient upregulation of ACTA2, DES, ADGRA2(GPR124), and FOXF2 (FIG. 3B). Expression levels of CSPG4, PDGFRB, CNN1,TAGLN, FOXF2, ABCC9, KCNJ8, DES, and ADGRA2 were similar in NCSC-derivedmural cells and primary brain pericytes; however, consistent with thelack of α-SMA expression (FIG. 2H; FIG. 8G,L), NCSC-derived mural cellsexpressed nearly 100-fold less ACTA2 transcript than primary pericytes(FIG. 3B). By D45, NCSC-derived mural cells retained expression of mostmarkers at levels similar to D25 cells, while ANPEP, ABCC9, and KCNJ8expression further increased, suggesting these cells may continue tomature during extended culture in E6+10% FBS (FIG. 3B). Comparison oftranscripts upregulated in NCSC-derived mural cells compared to theirNCSC precursors revealed several enriched Gene Ontology (GO) termsincluding vascular development, blood vessel morphogenesis, andextracellular matrix organization (FIG. 3C), indicating thatdifferentiation is driving the progression from NCSCs to mural cellswith vascular-associated transcript signatures. Of the 46 genes withhuman homologs identified as pericyte-enriched by single cell RNA-seq inmice (40), 29 were expressed at or above 1 FPKM by NCSC-derived muralcells and 26 by primary pericytes (FIG. 3D; Table 4). Finally, NCSCsmaintained in E6-CSFD retained neural crest marker expression and didnot develop expression of pericyte markers (FIG. 12 ). Collectively,these data demonstrate that differentiation of NCSCs in E6+10% FBSyielded a mural cell population that expressed pericyte-associatedmarkers while closely mimicking primary brain pericytes at atranscriptome level. Thus, we refer to the NCSC-derived mural cells asbrain pericyte-like cells throughout the remainder of the Example.

TABLE 4 Pericyte-enriched genes identified by single cell RNA-sequencingin mouse³⁹ with human homologs FPKM hPSC- Gene Primary derived* ABCC90.3 0.1 AGAP2 0.0 0.0 ANK2 9.6 2.3 ANO4 0.2 2.5 APOD 0.2 0.6 APOE 42.925.6 ARHGAP31 3.4 7.6 CORO1B 68.4 56.1 ECE1 58.7 31.0 EMCN 3.3 1.7FAM118B 0.0 0.0 FBLN1 24.7 112.0 FLT1 4.7 7.6 FOXF2 4.4 0.9 GGT1 104.8181.9 GPR4 5.4 4.6 IFI30 18.4 17.2 IGF2 0.3 14.0 ITIH5 2.6 0.2 ITM2A 0.33.9 JUP 6.4 20.8 KCNJ8 0.8 0.3 LGALS9 5.1 3.4 NODAL 0.1 0.1 NRXN2 2.50.8 NXPH4 30.3 0.4 PCDHGC3 0.0 0.0 PDE2A 0.6 0.1 PDE8A 6.5 6.0 PHC1 0.010.8 PHLDB1 28.5 28.8 PLOD1 189.1 145.8 POR 33.4 28.4 PPP1CC 40.7 82.8PREX2 0.0 0.0 SEPP1 (SELENOP) 19.8 1.4 SFRP2 0.0 16.7 SLC22A8 0.0 0.0SLC6A13 0.4 0.1 SPP1 0.96 11.6 ST8SIA4 0.0 0.3 SYNE1.2 (SYNE1) 16.9 6.0TNFRSF19 0.4 13.6 TRPC3 0.2 0.1 TTLL3 160.3 210.9 UCHL1 739.1 415.9Number with FPKM ≥1 26 29 *Average of all D25 hPSC-derived pericyte-likecell differentiations (H9 A-C, CS03n2 and IMR90C4)

Brain Pericyte-Like Cells Assemble with Vascular Cord Networks

Pericytes associate with endothelial cells and stabilize nascentvascular networks (51). To assess the ability of brain pericyte-likecells to self-assemble with endothelial cells, an in vitro endothelialcord forming assay was performed. A 3:1 mixture of primary pericytes orhPSC-derived brain pericyte-like cells (D22) and human umbilical veinendothelial cells (HUVECs) or immortalized human BMECs (hBMECs) wasplated on Matrigel (FIG. 4A; FIG. 13A). H9, CS03n2 and IMR90C4-derivedbrain pericyte-like cells self-associated with HUVECs and hBMECs muchlike primary human brain pericytes (FIG. 4B-C; FIG. 13B-C). After 24hours, hPSC-derived pericyte-like cells exhibited high NG2 expressionand aligned along the CD31⁺ endothelial cell cord perimeter anddeveloped pericyte-like morphology with stellate-shaped bodies andextended cell processes (FIG. 4B-C). Whereas HUVECs or hBMECs alone andHUVECs or hBMECs in co-culture with control HEK293 cells yielded manysmall branching cords, co-culture with the hPSC-derived brainpericyte-like cells or primary human brain pericytes yielded fewer,appreciably longer cords (FIG. 4C-F; FIG. 13C-F). These data demonstratethat hPSC-derived NCSC lineage mural cells exhibit pericyte-likeassociation with endothelial cells leading to the formation of more welldeveloped cord networks.

Brain Pericyte-Like Cells Induce Blood-Brain Barrier Properties

To investigate if hPSC-derived brain pericyte-like cells canrecapitulate key BBB inducing properties that have been observed invivo, such as reduction in tight junction abnormalities andtranscytosis, we next co-cultured the pericyte-like cells withhPSC-derived BMECs generated as we previously described (56). When D22brain pericyte-like cells were co-cultured with hPSC-derived BMECs, theBMEC barrier properties as measured by TEER were substantially elevated,while co-culture with a non-inducing cell type (3T3) yielded no barrierenhancement (FIG. 5A-B). TEER elevation by hPSC-derived brain-pericytelike cells was indistinguishable from that induced by primary humanbrain pericytes (FIG. 5B). The TEER increases were accompanied by acorresponding decrease in permeability to fluorescein, a hydrophilic,small molecule tracer (FIG. 5C). After BMEC co-culture, the brainpericyte-like cells remained NG2⁺/PDGFRβ⁺ (FIG. 10A), indicating theircontinued maintenance of mural identity. To determine tight junctionchanges that may drive the induction in BMEC barrier properties, theexpression level and localization of tight junction proteins occludinand claudin-5 were evaluated in the BMECs. Expression levels of occludinand claudin-5 were unchanged by co-culture (FIG. 10B). In addition,quantitative immunocytochemical evaluation of occludin and claudin-5indicated that the number of cells possessing continuous tight junctionswas unchanged upon treatment with pericyte-conditioned medium (FIG.5D-E; FIG. 10C). However, the percentage of cells with occludin tightjunction abnormalities or fraying was substantially reduced by treatmentwith pericyte-conditioned medium (FIG. 5F), correlating with the reducedpermeability, while claudin-5 fraying remained unchanged (FIG. 10C).

Next, the effects of brain pericyte-like cell co-culture on BMECtranscytosis properties were evaluated. To test non-specific molecularuptake and transcytosis in BMECs, a 10 kDa Alexa 488-tagged dextran wasdosed into the apical Transwell chamber and accumulation into andtranscytosis across the BMEC monolayer were quantified. After BMECculture with medium conditioned by hPSC-derived brain pericyte-likecells, confocal imaging indicated a qualitative decrease inintracellular dextran uptake in punctate vesicular structures, similarto that observed with primary human brain pericytes; whereas, mediumconditioned by 3T3 control cells had no effect (FIG. 10D). Indeed,quantification of dextran accumulation in BMECs co-cultured with brainpericyte-like cells or primary brain pericytes indicated that BMECaccumulation was reduced by about 30% (FIG. 5G). These differences inaccumulation translated to a corresponding 30% decrease in 10 kDadextran transcytosis upon pericyte co-culture (FIG. 5H). In contrast,when 10 kDa dextran transport was measured at 4° C., conditions thatsignificantly inhibit vesicular transcytosis processes, pericyteco-culture did not affect dextran transport compared to 3T3s orhPSC-derived BMEC monoculture (FIG. 51 ), indicating that the observeddecreases in dextran transport could not be ascribed to differences inparacellular transport resulting from improved tight junction fidelity.

Finally, to confirm that the effects of hPSC-derived brain pericyte-likecells are not specific to BMECs derived from hPSCs, the induction of BBBbarrier and transcytosis attributes was also evaluated in primary ratBMECs. Co-culture with IMR90C4-derived brain pericyte-like cellselevated the TEER in primary rat BMECs to the same level as observedwith primary human brain pericytes (FIG. 11A). In addition, co-culturewith brain pericyte-like cells also reduced accumulation andtranscytosis of 10 kDa dextran in primary rat BMECs (FIG. 11B,C). Insummary, these data indicate that hPSC-derived brain pericyte-like cellscan induce BBB phenotypes including elevation of BMEC barrier tightnessand reduction in transcytosis.

iPSC-Derived Brain Pericyte-Like Cells can be Integrated into anIsogenic NVU Model

Previously, we demonstrated that sequential co-culture of iPSC-derivedBMECs with primary pericytes and primary neural progenitor-derivedastrocytes and neurons enhanced BMEC barrier tightness (30).Subsequently, iPSC-derived astrocytes and neurons were shown to inducebarrier formation in iPSC-derived BMECs (34). Here, iPSC-derived brainpericyte-like cells were combined with iPSC-derived BMECs, astrocytes,and neurons to model the NVU. IMR90C4-derived BMECs were sequentiallyco-cultured with IMR90C4-derived brain pericyte-like cells andIMR90C4-derived astrocyte/neuron cultures (PNA) and compared toIMR90C4-derived BMEC monocultures or IMR90C4-derived BMECs co-culturedwith pericytes (P) or astrocytes/neurons (NA) alone (FIG. 6A). All threeco-culture conditions (P, NA, and PNA) significantly elevated TEER abovemonoculture (FIG. 6B). While neuron/astrocyte co-culture slightlyelevated TEER above pericyte co-culture (720±84 Ω·cm² NA co-culture vs.503±63 Ω·cm² P co-culture), the combination of pericyte andneuron/astrocyte co-culture treatments further elevated BMEC TEER(1156±94 Ω·cm² vs. NA and P co-culture) (FIG. 6B). All three co-cultureconditions yielded a five-fold reduction in sodium fluoresceinpermeability compared to monoculture conditions but no appreciabledifferences were observed between separate co-culture treatmentconditions (FIG. 6C), as has been reported previously for BMECmonolayers with TEER values exceeding ˜500-600 Ω·cm² (30, 34, 57). Thesedata demonstrate that iPSC-derived brain pericyte-like cells can bereadily combined with iPSC-derived BMECs, astrocytes and neurons to forman isogenic model of the human NVU.

Brain pericytes play essential roles in BBB formation and maintenance byregulating BMEC transcytosis, barrier fidelity, vascular structure andstability (5, 13, 14, 19-21). Here we report that mural cells can bedifferentiated from hPSC-derived NCSCs, and that these cells developbrain pericyte-like attributes. The brain pericyte-like cells canself-assemble with endothelial cells in vitro and impact their vascularnetwork structure. Moreover, the brain pericyte-like cells induce BBBproperties, including barrier tightening and reduction of transcytosisin BMECs. Finally, these cells can be incorporated into an isogeniciPSC-derived NVU model, with potential applications in patient-specificNVU modeling.

During embryonic development, NCSCs are first specified at the interfacebetween the neural plate and non-neural ectoderm, and subsequentlyreside in the dorsal neural tube before migrating throughout the embryoand differentiating to diverse cell types (58). Previous NCSCdifferentiation protocols have relied on differentiating hPSCs toneuroectoderm and subsequently isolating NCSC subpopulations (41, 47),or have used a directed WNT activation and activin/nodal inhibitionapproach to obtain NCSCs (46, 59, 60). We chose to utilize the latterapproach given its simplicity and potential for highly enriched NCSCpopulations. BMP signaling activation was previously shown to inhibitNCSC formation (46); however, the need to inhibit BMP signaling duringNCSC differentiation has been variable (42, 46). Here, when thedifferentiation strategy was adapted to minimal E6 medium, inhibition ofBMP signaling was necessary to efficiently direct hPSCs top75-NGFR⁺/HNK1⁺ NCSCs. The NCSCs differentiated in E6-CSFD medium were ahighly enriched population of multipotent cells having the capacity toform mesenchymal derivatives and peripheral neurons using multiple hPSClines.

A common approach to differentiate mural cells from NCSCs is tosupplement basal medium with PDGF-BB and TGFβ1 (41, 42, 47). Resultantcells have been shown to express calponin, SM22α, and α-SMA (41, 42,47), but two key mural cell markers, PDGFRβ and NG2 were not previouslyexamined. While differentiation of NCSCs in E6 medium yielded PDGFRβ⁺cells, neither E6 medium nor E6 medium supplemented with PDGF-BB andTGFβ1 generated cells expressing NG2. However, both calponin and SM22αwere expressed even in the absence of growth factor supplementation.Instead, when E6 was supplemented with 10% FBS, the differentiatingcells acquired NG2 and PDGFRβ expression, and thus were classified asforebrain lineage mural cells (40, 49). Recent work demonstratedpericyte differentiation from hPSC-derived cranial neural crest cellsusing PDGF-BB, however 2-5% FBS was included in the differentiationmedium (37). Thus, it is possible that the observed pericytedifferentiation is mediated at least partially by FBS, consistent withour observations. Alternatively, the requirement of PDGF-BB may reflectdifferences in initial neural crest phenotypes or basal media. Whileothers have suggested the use of serum to drive mural celldifferentiation from NCSC (46, 47), these studies generated cells thatwere smooth muscle actin positive. In contrast, we did not observesubstantial α-SMA expression in the differentiated mural cells, evenafter extended culture. Brain pericytes lining higher order capillariesgenerally do not express α-SMA in vivo (40, 61, 62), although veryrecent evidence suggests that higher order pericytes may actuallyexpress low levels of α-SMA that are lost upon sample preparation (63).In addition, it is well known that upon fresh isolation, primary brainpericytes express α-SMA in 5-10% of cells, whereas after a few days inculture they become nearly uniformly α-SMA+(38, 39), as also observedhere with primary human brain pericytes, which expressed α-SMA. Thus,the lack of α-SMA expression in the differentiated brain pericyte-likecells better reflects the lack of α-SMA in brain pericytes in vivo.However, much like primary brain pericytes and previous reports withNCSC derived mural cells, we observed sustained expression of thecontractile-related proteins calponin and SM22α. In addition,differentiation of mesenchymoangioblasts towards pericyte lineagesyielded cells that expressed differential levels of calponin (64).Although SM22α is an early developmental marker of mural cells (65), arecent single cell transcriptomics study strongly suggests that murinebrain capillary pericytes in vivo do not express calponin-encoding Cnn1or SM22α-encoding Tagln (40). Additional transcript evaluation confirmedthe brain signature of the pericyte-like cells (ZIC1, FOXF2) (54, 55).The brain pericyte-like cells also expressed transcripts for ABCC9 andKCNJ8, two additional markers that differentiate brain capillarypericytes from other mural cell types (40, 49), and these markers werefurther elevated over extended culture times. RNA-sequencing alsoindicated a transcriptome-wide similarity to primary human brainpericytes and expression of many genes identified as pericyte-enrichedby single cell RNA-sequencing in mouse (40). Taken together, thehPSC-derived brain-pericyte like cells had marker profiles thatsuggested the generation of cells similar to brain pericytes.

While the marker expression suggested that the differentiation processgenerated brain pericyte-like cells, it is most important that the cellsrecapitulate key functional attributes of brain pericytes. When culturedwith HUVECs or hBMECs, brain pericyte-like cells aligned along vascularcords and extended cell processes. Primary pericytes can stabilizeendothelial cell cord formation in vitro (26). This phenotype was alsoobserved with both primary human brain pericytes and hPSC-derived brainpericyte-like cell co-culture as indicated by reduced numbers of longercords as has also been reported using hPSC-derived pericytes ofmesenchymal origins (64). In addition to this more general pericytephenotype, it was expected that a brain pericyte-like cell would impactthe barrier and non-specific transcytosis properties of brainendothelial cells (13, 14). Indeed, BBB properties of both hPSC-derivedBMECs and primary rat BMECs were substantially induced by co-culturewith hPSC-derived brain pericyte-like cells, and these effects mimickedthose induced by primary human brain pericytes. TEER was increasedsubstantially as expected (13, 30). Correlating with this increasedbarrier function, pericyte co-culture decreased the number of frayedoccludin tight junctions as seen previously for a variety of barrierinductive stimuli (29, 34, 66), but did not alter the expression levelsof tight junction proteins occludin or claudin-5. These results mirrorthose in vivo where tight junction structure was altered by pericytesalthough the expression levels of tight junction proteins were notaffected (13). We also demonstrated that non-specific cellularaccumulation and transcytosis were downregulated in BMECs afterco-culture with brain-pericyte like cells, and the effects wereindistinguishable from those elicited by primary human brain pericytes.These phenotypes combined with the developmental origins and markerexpression profile, along with the similarities to primary human brainpericytes, suggest that we have generated a novel hPSC-derived cell thatcan model human brain pericytes.

While many studies have utilized primary brain pericytes to enhance BMECbarrier properties, primary brain pericytes offer limited scalability,especially for human in vitro BBB models (28, 30, 67). In addition,limited primary cell availability essentially eliminates the possibilityof using patient matched brain pericytes and BMECs that could be usedfor disease modeling applications. Here, we demonstrate the capabilityto differentiate brain like pericytes in a scalable fashion (˜1000 brainpericyte-like cells per input stem cell). Moreover, the differentiationis reproducible amongst differentiations and across hPSC lines. Thus,the ability to derive brain pericyte-like cells from patient-derivediPSCs provides a unique tool for the study of patient-specific pericytecontributions to CNS disorders that have been suggested to have pericyteinvolvement such as stroke, epilepsy, demyelinating disease, andAlzheimer's disease (7, 10, 68-71). In addition, lineage-specificdifferences have been noted in hPSC-derived pericytes, motivating theuse of pericytes from appropriate developmental origins for diseasemodeling applications (41). The brain pericyte-like cells can also beused in multicellular NVU models to capture the cellular crosstalk thatis likely responsible for many disease processes at the BBB. To thisend, we have demonstrated that it is possible to generate BMECs,neurons, astrocytes, and brain pericyte-like cells from a single iPSCcell line and combine them to form an isogenic NVU model having optimalTEER. These findings followed similar trends to our earlier reportswhere iPSC-derived BMEC properties were enhanced by co-culture withbrain pericytes and neural progenitor cell-derived astrocytes andneurons from primary sources. We note, however, that thisTranswell-based model lacks the potentially important contributions ofcell-cell contact and fluid shear stress, motivating future efforts tointegrate hPSC-derived NVU cell types into microfluidic or cellaggregate-based in vitro models (72, 73). It is likely that thesemulticellular NVU models will be used to uncover new mechanisms of BBBregulation in health and disease and assist in the therapeuticdevelopment process for CNS disorders.

MATERIALS AND METHODS hPSC Maintenance

IMR90C4 and CS03n2 iPSCs and H9 hESCs were maintained on Matrigel coatedplates in E8 medium, which is DMEM/F12 basal medium supplemented withL-ascorbic acid-2-phosphate magnesium (64 mg/L), sodium selenium (14μg/L), FGF2 (100 μg/L), insulin (19.4 mg/L), NaHCO₃ (543 mg/L),transferrin (10.7 mg/L), and TGFβ1 (2 μg/L) and prepared according toChen et al. (74). When cells reached ˜70% confluence, cells werepassaged using Versene to new Matrigel coated plates. For hPSCs used inBMEC differentiations, cells were maintained in mTeSR1 on Matrigelplates and passaged as previously described (75).

NCSC Differentiation

One day prior to initiating NCSC differentiation, hPSCs maintained in E8medium were singularized using Accutase and seeded at 9.1×10⁴ cells/cm²onto Matrigel coated plates with E8+10 μM Y27632. NCSC differentiationwas initiated the next day by switching medium to E6, which is DMEM/F12basal medium supplemented with L-ascorbic acid-2-phosphate magnesium (64mg/L), sodium selenium (14 μg/L), insulin (19.4 mg/L), NaHCO₃ (543mg/L), and transferrin (10.7 mg/L). E6 was supplemented with 22.5 mg/Lheparin sodium salt from porcine mucosa to stabilize FGF2, 1 μMCHIR99021, 10 μM SB431542 (Tocris), 10 μg/L FGF2, and 1 dorsomorphin,hereafter labeled E6-CSFD. Cells were expanded by replacing E6-CSFDdaily and passaging cells every time cells reached 100% confluence tofresh Matrigel coated plates. During passaging, cells were singularizedusing Accutase and replated at a splitting density of one 6-well to 6new 6-wells in E6-CSFD medium. Cells were generally passaged without 10μM Y27632. However, to increase IMR90C4 cell line survival during firstpassaging following NCSC differentiation initiation, IMR90C4 cells werereplated in E6-CSFD+10 μM Y27632. Subsequent IMR90C4 NCSC expansionpassages were replated without Y27632. Cells were typically passaged 2-3days following NCSC differentiation initiation and subsequently passagedevery 3-6 days depending on cell growth kinetics.

Magnetic Activated Cell sorting of NCSCs

At D15 of E6-CSFD treatment, cells were dissociated using Accutase andlabeled with 20 μL/10⁷ cells NCSC microbeads (Miltenyi), 20 μL/10⁷ cellsFcR blocking reagent, and 60 μL/10⁷ MACS buffer (0.5% BSA+2 mM EDTA insterile PBS without Ca²⁺/Mg²⁺) at 4° C. for 15 minutes. Cells werewashed in MACS buffer and resuspended in 500 μL MACS buffer/2×10⁷ cells.Cells were sorted through two LS columns (Miltenyi) according tomanufacturer instructions and resuspended in E6-CSFD+10 μM Y27632 toappropriate density for specific NCSC lineage differentiations asdescribed below.

NCSC Lineage Differentiations

For differentiation of peripheral neurons, after MACS sorting,hPSC-derived NCSCs were replated on Matrigel-coated plates and expandedfor 14 days in E6-CSFD. These cells were replated on Matrigel-coated12-well plates at 5×10⁴ cells/cm² in E6-CSFD. The following day, themedium was switched to peripheral neuron medium composed of DMEM/F12,1×N2 supplement, 10 ng/ml BDNF, 10 ng/ml GDNF, 10 ng/ml NT-3, 10 ng/mlNGF-β, 200 μM ascorbic acid (AA), and 0.5 mM cAMP, and replaced every 2days for 2 weeks.

For differentiation of mesenchymal derivatives, after MACS sorting,hPSC-derived NCSCs were replated on noncoated polystyrene plates andexpanded for 11 days in E6-CSFD. For adipogenesis, expanded hPSC-derivedNCSCs were seeded at a density of 10,000 cells/cm² and treated withadipogenic medium composed of high-glucose DMEM, 10% FBS, 1%antibiotics, 1 μg/ml insulin, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX),and 1 μM dexamethasone (Sigma-Aldrich). For osteogenesis, the seedingdensity was 5,000 cells/cm² and the cells were treated with osteogenicmedium consisting of low-glucose DMEM, 10% FBS, 1% antibiotics, 50 μg/mlAA, 10 mM β-glycerophosphate, and 0.1 μM dexamethasone. Forchondrogenesis, 250,000 NCSC were collected to form a high cell densitypellet by centrifuged at 600 g for 5 minutes and treated withchondrogenic medium containing high-glucose DMEM, 1% antibiotics and ITSPremix (BD Bioscience), 40 μg/ml L-proline, 50 μg/ml AA, 0.9 mM sodiumpyruvate (Sigma-Aldrich), 0.1 μM dexamethasone, and 10 ng/ml of freshlyadded transforming growth factor β1 (TGFβ1) (Peprotech). Medium waschanged every 3 days for all three differentiation procedures.

To analyze adipogenic differentiation, cells were fixed in 4% offormaldehyde and stained with Oil Red O (Sigma-Aldrich) for lipiddroplet formation. To analyze osteogenic differentiation, cells werefixed in 60% isopropanol and stained with Alizarin Red (RowleyBiochemical, Danvers, MA, USA) to evaluate mineral deposition.Chondrogenic potential was assessed by Alcian blue staining. Cellpellets were first fixed in 4% formaldehyde for 2 hours. Next, the cellpellet was dehydrated by a series of increasing concentration ofethanol, infiltrated with xylene, and then embedded with paraffin.Embedded cell pellets were cut into 8 μm sections using a microtome andstained with Alcian blue (Polysciences, Warrington, PA, USA) todetermine the glycosaminoglycan (GAG) content.

Pericyte Differentiation Factor Identification

Following MACS sorting, NCSC were replated onto 48-well plates inE6-CSFD medium+10 μM Y27632. Cells were switched to mural celldifferentiation medium the next day, expanded for six days, and stainedfor NG2/PDGFRβ expression. Cells were expanded on uncoated plates in E6medium, E6 medium supplemented with 2 ng/mL TGFβ1+20 ng/mL PDGF-BB, orE6 medium supplemented with 10% fetal bovine serum (FBS). Cells werealso expanded in E6 supplemented with 10% FBS on gelatin-coated platesprepared by coating plates for at least 1 h at 37° C. with a 0.1%gelatin A solution dissolved in water.

Immunocytochemistry

Cells were fixed fifteen minutes at room temperature with either 4%paraformaldehyde (PFA) or 100% ice-cold methanol depending on antibodystaining conditions. Cells were rinsed three times in PBS withoutCa²⁺/Mg²⁺ and stored at 4° C. in PBS until ready to stain. Afteraspirating PBS, cells were blocked one hour in blocking buffer at roomtemperature and incubated overnight at 4° C. on a rocking platform withprimary antibodies diluted in primary antibody staining buffer.Antibodies and staining conditions are listed in Table 2. The followingday, cells were washed three times with PBS and incubated with secondaryantibodies diluted 1:200 in primary antibody staining buffer. Cells wereprobed one hour in the dark at room temperature on a rocking platform.Afterwards, secondary antibody staining buffer was aspirated, and cellswere incubated five minutes with 4 μM Hoechst 33342 diluted in PBS.Cells were washed three times with PBS and stored at 4° C. in PBS in thedark until ready to image. Images were taken on Olympus epifluorescenceand Nikon A1R-Si+confocal microscopes.

TABLE 2 Antibody Staining Conditions Vendor (Clone/Catalog #, MarkerFixative App. Species) Dilution Blocking Buffer Staining Buffer p75-NGFR4% PFA ICC Advanced Targeting 1:1000 1% BSA 1% BSA Systems (ME20.4,Mouse IgG₁) Flow Advanced Targeting 0.2 μL/10⁶ cells N/A PBS Systems(ME20.4, Mouse IgG₁) HNK1 4% PFA ICC Sigma (C6608, 1:300 1% BSA 1% BSAMouse IgM) Flow Sigma (C6608, 0.2 μL/10⁶ cells N/A PBS Mouse IgM) AP2 4%PFA ICC DSHB (3B5, Mouse 1:50 1% BSA + 0.1% 1% BSA IgG_(2B)) TX-100Peripherin 4% PFA ICC Millipore (AB1530, 1:200 1% BSA + 0.1% 1% BSARabbit Polyclonal) TX-100 βIII-Tubulin 4% PFA ICC Sigma (T8860, 1:500 1%BSA + 0.1% 1% BSA Mouse IgG_(2b)) TX-100 NG2 4% PFA ICC Millipore(MAB2029, 1:100 5% goat serum + PBS + 0.4% Mouse IgG_(2a)) 0.4% TX-100TX-100 Flow Millipore (MAB2029, 2 μL/10⁶ cells N/A MACS Buffer MouseIgG_(2a)) PDGFRβ 4% PFA ICC Cell Signaling 1:100 5% goat serum + PBS +0.4% Technology (28E1, 0.4% TX-100 TX-100 Rabbit Monoclonal) Flow BDBiosciences 1.25 μL/10⁶ cells N/A MACS Buffer (28D4, Mouse IgG_(2a))CNN1 4% PFA ICC Sigma (C2687, 1:15000 3% BSA + 0.1% 3% BSA Mouse IgG₁)TX-100 SM22α 4% PFA ICC Abcam (ab14106) 1:1000 3% BSA + 0.1% 3% BSATX-100 CD31 4% PFA ICC Thermo Fisher (RB- 1:25 5% goat serum + PBS +0.4% 10333, Rabbit 0.4% TX-100 TX-100 Polyclonal) aSMA 4% PFA ICC LabVision (MS-113- 1:100 5% milk + 0.1% 5% milk P) TX-100 β-actin N/A WBCell Signaling 1:1000 TBST + 5% milk TBST + 5% milk Technology (13E5,Rabbit Monoclonal) Occludin MeOH ICC Invitrogen (OC- 1:200 10% goatserum 10% goat serum 3F10, Mouse IgG₁) N/A WB Invitrogen (OC- 1:500TBST + 5% milk TBST + 5% milk 3F10, Mouse IgG₁) Claudin-5 N/A WBInvitrogen (4C302, 1:250 TBST + 5% milk TBST + 5% milk Mouse IgG₁) CD134% PFA ICC R&D Systems 1:50 10% goat serum 10% goat serum (MAB3815,Mouse IgG_(2a)) Desmin MeOH ICC Thermo Fisher (RB- 1:50 10% goat serum10% goat serum 9014, Rabbit Polyclonal)

Flow Cytometry

Cells were incubated 30 minutes on ice with primary antibody diluted in100 μL/sample primary antibody staining buffer as indicated in Table 2.Cells were washed one time with cold PBS (p75-NGFR/HNK1 flow cytometry)or MACS buffer (NG2 and PDGFRβ flow cytometry). Cells were subsequentlyincubated in 100 μL primary antibody staining buffer with 1:500Alexa-tagged isotype-specific goat secondary antibodies. Cells werewashed as previously described and resuspended in 4% PFA for 15 minutesat room temperature. Cells were subsequently stored in wash buffer forup to 24 hours at 4° C. prior to running samples on cytometer.

Temporal RNA Analysis

Cells were harvested using Accutase, quenched in DMEM/F12, and spun down5 minutes at 200 g. After removing the supernatant, cell pellets weresnap frozen at −80° C. until ready for mRNA extraction. The RNeasy MiniKit (Qiagen) was used to extract mRNA, including a cell lysatehomogenization step on QIAshredder Columns (Qiagen), according tomanufacturer instructions. DNA was removed on column using theRNase-free DNase Set (Qiagen). Extracted RNA was stored in nuclease-freewater at −20° C. until ready to reverse transcribe to cDNA. RNA wasreverse transcribed at a concentration of 250 ng/mL into cDNA usingOmniscript reverse transcriptase kit (Qiagen) and Oligo(dT)₂₀ Primers(Life Technologies). Temporal gene expression analysis was conductedusing 25 μL PCR reactions containing GoTaq Green Master Mix (Promega),10 ng/reaction cDNA template, and 100 nM forward/reverse primers. PCRwas run according to manufacturer protocols, and all reactions includeda no template and mRNA control to verify no genomic DNA contamination oramplification. PCR primer sequences, annealing temperatures, and cycletimes are listed in Table 3. PCR products were resolved on a 2% agarosegel, stained using ethidium bromide, and imaged on a ChemiDoc XRS+System(Bio-Rad).

TABLE 3 DNA Probe Sequences and Running Conditions T_(a) Gene (° C.)Cycles Fwd Sequence Rev Sequence ABCC9 60 405′-TCA ACC TGG TCC CTC ATG TCT-3′ 5′-CAG GAG AGC GAA TGT AAG AAT CC-(SEQ ID NO: 1) 3′ (SEQ ID NO: 2) ACTA2 60 305′-TGT TCC AGC CAT CCT TCA TC-3′ 5′-GCA ATG CCA GGG TAC ATA GT-(SEQ ID NO: 3) 3′(SEQ ID NO: 4) ANPEP 49 405′-GAA GAG AAC TGG AGG AAG ATT CAG- 5′-CCA GGT TGA AGG CGT CAT TA-3′3′ (SEQ ID NO: 5) (SEQ ID NO: 6) B3GAT1 58 355′-TCG CCT GGA CTG GAC TGG GG-3′ 5′-TGG CCT TGG CCT CCC TCC TC-3′(SEQ ID NO: 7) (SEQ ID NO: 8) CNN1 53 405′-GTC CAC CCT GGC TTT-3′ (SEQ ID 5′-AAA CTT GTT GGT GCC CAT CT-3′NO: 9) (SEQ ID NO: 10) CSPG4 54 35IDT DNA Hs.PT.58.39417158 Predesigned Probe FOXF2 52 405′-ACC AGA GCG TCT GTC AGG ATA TT- 5′-GTG ACT TGA ATC CGT CCC AGT TTC-3′ (SEQ ID NO: 11) 3′ (SEQ ID NO: 12) GAPDH 60 305-GAA GGT GAA GGT CGG AGT CAA CG- 5′-TCC TGG AAG ATG GTG ATG GGA T-3′3′ (SEQ ID NO: 13) (SEQ ID NO: 14) KCNJ8 60 405′-GTG ATT GCC GTC CGA AAT GG-3′ 5′-AGT TGG TGA ATA GGA ACC ACC T-3′(SEQ ID NO: 15) (SEQ ID NO: 16) NANOG 58 305′-CGA AGA ATA GCA ATG GTG TGA CG- 5′-TTC CAA AGC AGC CTC CAA GTC-3′3′ (SEQ ID NO: 17) (SEQ ID NO: 18) NGFR 60 305′-GTG GGA CAG AGT CTG GGT GT-3′ 5′-AAG GAG GGG AGG TGA TAG GA-3′(SEQ ID NO: 19) (SEQ ID NO: 20) PDGFRB 53 405′-GCT CAC CAT CAT CTC CCT TAT C-3′ 5′-CTC ACA GAC TCA ATC ACC TTC C-3′(SEQ ID NO: 21) (SEQ ID NO: 22) POU5FI 58 305′-CAG TGC CCG AAA CCC ACA C-3′ 5′-GGA GAC CCA GCA GCC TCA AA-3′(SEQ ID NO: 23) (SEQ ID NO: 24) SOX10 61 405′-ATA CGA CAC TGT CCC GGC CCT AAA-5′-TTC TCC TCT GTC CAG CCT GTT CTC-3′ 3′ (SEQ ID NO: 25) (SEQ ID NO: 26)SOX9 60 40 5′-AGC GAA CGC AACA TCA AGA C-3′5′-CTG TAG GCG ATC TGT TGG GG-3′ (SEQ ID NO: 27) (SEQ ID NO: 28) TAGLN51 40 5′-TCT TTG AAG GCA AAG ACA TGG-3′ 5′-TTA TGC TCC TGC GCT TTC TT-3′(SEQ ID NO: 29) (SEQ ID NO: 30) TBX18 60 405′-CCC AGG ACT CCC TCC TAT GT-3′ 5′-TAG GAA CCC TGA TGG GTC TG-3′(SEQ ID NO: 31) (SEQ ID NO: 32) TFA2AP 50 305′-TCC CTG TCC AAG TCC AAC AGC AAT- 5′-AAA TTC GGT TTC GCA CAC GTA CCC-3′ (SEQ ID NO: 33) 3′ (SEQ ID NO: 34) ZIC1 59 405′-TGG CCC GGA GCA GAG TAA T-3 (SEQ 5′-CCC TGT GTG CGT CCT TTT G-3′ID NO: 35) (SEQ ID NO: 36)

RNA-Sequencing

RNA was extracted from H9 hESCs, H9-derived NCSCs at D15, H9-derivedNCSCs maintained for 40 additional days in E6-CSFD (D55), H9-derivedpericyte-like cells at D19, D22, and D25 (three independentdifferentiations at the D25 time point), H9-derived pericyte-like cellsmaintained for 20 additional days in E6+10% FBS (D45), CS03n2-derivedpericyte-like cells at D25, IMR90C4-derived pericyte-like cells at D25,and primary brain pericytes using the RNeasy Mini Kit (Qiagen) asdescribed above. TruSeq stranded mRNA libraries were prepared, cDNAsynthesized, pooled, and distributed over two sequencing lanes, andsamples sequenced on an Illumina HiSeq 2500 by the University ofWisconsin-Madison Biotechnology Center. Reads were mapped to the humangenome (hg38) with HISAT2 (v2.1.0) and transcript abundances (fragmentsper kilobase of transcript per million mapped reads, FPKM) quantifiedwith Cufflinks (v2.1.1). FPKM values from the two sequencing lanes foreach sample were averaged. Hierarchical clustering was performed withMorpheus (software.broadinstitute.org/morpheus) using the one minusPearson correlation with average linkage. Gene ontology (GO) analysiswas performed using the PANTHER (76) online tool (pantherdb.org).

Matrigel Cord Formation Assay and Quantification

HEK293 fibroblasts and human umbilical vein endothelial cells (HUVECs)were maintained on tissue culture polystyrene flasks in DMEM+10% FBS.Immortalized human BMECs (hBMECs (77), a gift of Kwang Sik Kim andMonique Stins, Johns Hopkins University, Baltimore, MD) were maintainedin RPMI1640+10% FBS+10% NuSerum+lx MEM non-essential amino acids onflasks that had been coated with a solution of 1% rat tail collagen in70% ethanol that was allowed to evaporate. 8-well glass chamber slideswere coated with 200 μL/well concentrated growth factor reduced Matrigeland incubated at least one hour at 37° C. to set the Matrigel. HUVECs orhBMECs were plated at 2.2×10⁴ cells/8-well chamber slide in 500 μL EGM-2medium (Lonza) alone, with 6.6×10⁴ HEK293 fibroblasts, primary brainpericytes, or hPSC- derived mural cells at D22 of the differentiation.Cells were incubated 24 hours at 37° C. to allow cord formation andbright field images taken on live cells at 24 hours following plating.Cords were subsequently fixed and stained according theimmunocytochemistry methods listed above. Matrigel-associated cords weremounted onto glass slips and imaged using Olympus epifluorescence andNikon A1R-Si+confocal microscopes. Cord length and number of cords perfield were quantified by hand using the ImageJ ROI Manager Tool andaveraged over at least 3 independent fields per condition perdifferentiation.

BMEC Differentiation

IMR90C4 iPSCs were maintained in mTesR1 medium on Matrigel-coated platesand passaged as previously described. Three days prior to initiating adifferentiation, cells were seeded at 9×10⁴−10⁵ cells/cm² onto Matrigelcoated plates in mTeSR1+10 μM Y27632. Medium was replaced daily untilcells reached>2.6×10⁵ cells/cm². Cell medium was replaced withUnconditioned Medium (UM), containing 392.5 mL DMEM/F12, 100 mL KOSR(Gibco), 5 mL 100×MEM non-essential amino acids (Gibco), 2.5 mL100×Glutamax (Gibco), and 3.5 μL β-mercaptoethanol. Cells were replaceddaily with UM for six days, and subsequently switched to EC medium,containing hESFM+1% platelet-poor plasma derived serum (PDS), and 20ng/mL FGF2. Cells were incubated two days with EC medium withoutreplacing medium. Cells were sub-cultured at D8 onto 4:1:5collagen/fibronectin/water-coated Transwells or 5×diluted 4:1:5collagen/fibronectin/water-coated plates as detailed by Stebbins et al.(75). Cell culture medium was replaced with EC without FGF2 24 hoursafter subculturing hPSC-derived BMECs onto filters.

BMEC/Pericyte Co-Culture

Primary brain pericytes, hPSC-derived pericyte-like cells, and 3T3s wereseparately seeded onto poly-L-lysine coated 12-well plates (primarybrain pericytes) or uncoated plates (hPSC-derived early mural cells and3T3s) when early mural cells reached first reached 80-100% confluence,typically 3-4 days after initiating serum treatment on hPSC-derived NCSC(D19-D20). Cells were plated on the same day at equivalent seedingdensities of 5×10⁴ cells/12-well in either DMEM+10% FBS (primary brainpericytes and 3T3s) or E6+10% FBS (hPSC-derived pericyte-like cells).Cells were dissociated with either 0.25% Trypsin/EDTA (primary brainpericytes and 3T3s) or Accutase (hPSC-derived pericyte-like cells).hPSC-derived brain pericyte-like cell medium was replaced daily withE6+10% FBS until D22 of the differentiation. Pericytes and 3T3s were fedwith DMEM+10% FBS every two days until D22. At D22, cells were replacedwith 1.5 mL EC medium above 12-well polystyrene transwell filters with a0.4 μm pore size.

IMR90C4 iPSC-derived BMECs at D8 of the BMEC differentiation weresub-cultured onto Transwell filters at 1.1×10⁶ cells/12-well filter aspreviously described (75). The high seeding density is intended toensure a confluent monolayer suitable for TEER and permeabilitymeasurements. Cells were incubated two days in co-culture, with cellculture medium replaced at 24 hours after initiating co-culture with ECmedium without FGF2. Transendothelial electrical resistance (TEER) wasmeasured every 24 hours after initiating co-culture. 48 hours followingco-culture, BMEC Transwell filters were transferred to a fresh 12-wellplate for sodium fluorescein assays. Cell medium was replaced with 1.5mL of EC medium without FGF2 in the basolateral chamber and 0.5 mL ofthe same medium with 10 μM sodium fluorescein in the apical chamber.Cells were incubated one hour on a rotating platform and basolateralchamber medium collected every 15 minutes during the hour incubationperiod. After 1 hour, cell culture medium for the apical chamber wascollected to calculate sodium fluorescein permeability across BMECmonolayers following 48 hours of co-culture treatment. Fluorescenceintensity was measured using a Tecan plate reader set to a 485 nmexcitation and 530 nm emission settings. Permeability calculations weredetermined according to Stebbins et al. (75).

Transcytosis and Accumulation Assays

Following 48 hours of co-culture, BMEC-seeded transwells weretransferred to an empty plate. We utilized a 10 kDa dextran tagged withAlexa-488 to quantify the level of intracellular accumulation andtranscytosis. 10 μM dextran was suspended in 0.5 mL of EC medium withoutFGF2 onto the apical side of the transwell. To determine the level oftranscytosis, following two hours of incubation in a 37° C. incubator(20% O₂, 5% CO₂) on a rotating platform, we collected 150 μL from the1.5 mL of EC medium on the basolateral side of the transwell.Fluorescence intensity was measured using a Tecan plate reader set to a495 nm excitation and 519 nm emission settings. To determine the levelof accumulation (endocytosis) in the BMECs, we rinsed the transwellswith cold PBS (2×) and lysed the cells with radioimmunoprecipitationassay (RIPA) buffer. Lysates were collected and analyzed on the platereader. Fluorescence values were normalized to protein content/Transwellmeasured using the bicinchoninic acid (BCA) assay.

Tight Junction Image Analysis

BMECs were plated on 24-well plates in EC medium or EC mediumconditioned by primary brain pericytes or hPSC-derived pericyte-likecells. After 48 h, BMECs were fixed and stained for occludin asdescribed above. Images were acquired from 5 wells per experimentalcondition. To quantify tight junction continuity, images were blindedand the area fraction index determined using FIJI as previouslydescribed (78). Additionally, images were blinded and the number offrayed junctions (FIG. 4D) manually counted.

Isogenic Model of the Neurovascular Unit

BMECs were differentiated from iPSCs as previously described.Singularized BMECs were seeded onto collagen IV/fibronectin coatedtranswells at day 8 of the differentiation. hPSC-derived pericytes wereseeded onto the bottom of the co-culture plate (˜50,000 cells/cm²) in ECmedium. We additionally investigated the cumulative effects ofpericytes, neurons, and astrocytes. Neurons and astrocytes weredifferentiated from iPSCs as previously published (34) . Initially,BMECs were placed in co-culture with pericytes for 24 hours in EC mediumand then BMEC-Transwells were transferred to a co-culture plate with amixture of neurons and astrocytes (1:3 ratio) for the duration of theexperiment in EC medium without FGF2. We benchmarked our stemcell-derived BBB model (BMECs, pericyte-like cells (24h), and neuronsand astrocytes (24h)) to a BBB co-culture model absent of pericyte-likecells (neurons and astrocytes only). TEER and sodium fluoresceinpermeability assays were conducted on BMEC-Transwells.

Statistics

All experiments were conducted using at least three technical replicates(e.g. three 6-wells or Transwells) from the same differentiation. Allexperiments were replicated (independent differentiations) at leastthree times except where otherwise indicated. Data are presented asmean±SD of technical replicates from a representative differentiation oras mean±SEM of pooled data from several independent differentiations.Statistical significance was evaluated using one-way analysis ofvariance (ANOVA) followed by post-hoc tests controlling for multiplecomparisons: Dunnett's test for comparison of experimental groups tocontrol, and Tukey's test for comparison between all experimentalgroups. P<0.05 was considered statistically significant.

Western Blotting

BMECs were cultured on Transwells alone or co-cultured with hPSC-derivedpericyte-like cells, primary brain pericytes, or 3T3s as previouslydescribed. After 48 h of co-culture, BMECs were washed once with PBS andlysed with RIPA buffer+Halt protease inhibitor cocktail. The BCA assaywas used to determine protein concentration. Proteins were resolved on4-12% Tris-glycine gels and transferred to nitrocellulose membranes,which were blocked in Tris-buffered saline+0.1% Tween-20 (TBST)+5%nonfat dry milk for 1 h, and incubated with primary antibodies (Table 2)overnight at 4° C. Membranes were washed with TBST (5×) and incubatedwith donkey anti-mouse or donkey anti-rabbit IRDye 800CW secondaryantibodies (LI-COR) for 1 h. Membranes were washed with TBST (5×) andimaged using a LI-COR Odyssey.

Visualization of Dextran Accumulation

iPSC-derived BMECs were seeded onto glass bottom plates at a density of10⁵ cells/cm² and cultured for 48 h in EC medium or EC mediumconditioned by 3T3s, primary pericytes, or IMR90C4-derived pericyte-likecells. Medium was subsequently replaced with EC medium+10 μM Alexa488-tagged 10 kDa dextran. Following 2 h of dextran incubation, cellswere fixed in 4% PFA for fifteen minutes, followed by three washes inPBS. Cells were blocked in 10% goat serum in PBS for 30 minutes at roomtemperature. Cells were incubated with Anti-Alexa 488 antibody (1:100 inPBS; Life Technologies 11094) overnight at 4° C. on a rocking platform.Following three washes in PBS, cells were incubated with Alexa 647secondary antibody (1:200 in PBS) for one hour at room temperature on arocking platform. Nuclei were labeled with Hoechst and cells were rinsedthree times in PBS. Cells were visualized on a Nikon A1R-Si+confocalmicroscope. The lack of permeabilization allows internalized dextran tovisualize only with Alexa 488, while extracellular (surface) dextran isalso labeled with Alexa 647.

Primary Rat BMEC/Pericyte Co-Culture

All animal studies were conducted using protocols approved by theUniversity of Wisconsin-Madison Animal Care and Use Committee followingNIH guidelines for care and use of laboratory animals. Adult maleSprague-Dawley rat (Harlan Inc., Indianapolis, IN) brain capillarieswere isolated, minced and digested in collagenase type-2 (0.7 mg/mL) andDNase I (39 U/mL). Purified microvessels were isolated followingcentrifugation in 20% bovine serum albumin and further digested incollagenase/dispase (1 mg/mL) and DNase I. To purify the population a33% Percoll gradient was utilized. Capillaries were collected and platedonto collagen IV/fibronectin-coated Transwells. Capillaries werecultured in DMEM supplemented with 1 ng/mL FGF2, 1 μg/mL heparin, 20%PDS, 2 mM L-glutamine, and 1% antibiotic-antimitotic solution. Purepopulations were obtained by treating the cells with puromycin (4 μg/mL)for 2 days following seeding. Four days following isolation, ratBMEC-seeded Transwells were transferred onto plates containingIMR90C4-derived pericyte-like cells, primary brain pericytes, or 3T3s(described previously) and co-cultured in EC medium containing 1% PDS.

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1. A population of brain pericyte-like cells, wherein the cells expresspericyte markers but do not express actin alpha 2 (ACTA2), and whereinthe cells are generated from hPSCs.
 2. The population of cells of claim1, wherein the pericyte markers include calponin 1 (CNN1), neuron-glialantigen 2 (NG2), and platelet-derived growth factor receptor beta(PDGFRB).
 3. The population of brain pericyte-like cells of claim 1,wherein the population is at least 90% NG2⁺PDGFRB⁺.
 4. The cells ofclaim 3, wherein the brain pericyte-like cells further express calponinand smooth muscle 22α (SM22α) but do not express alpha-smooth muscleactin (α-SMA).
 5. The cells of claim 1, wherein the cells are capable ofassociating with vascular networks.
 6. The cell population of claim 1,wherein the cells express one or more transcripts of pericyte markersselected from the group consisting of chondroitin sulfate proteoglycan 4(CSPG4), PDGFRB, CNN1, transgelin (TAGLN), alanyl aminopeptidase(ANPEP), T-box transcription factor 18 (TBX18), ATP binding cassettesubfamily C member 9 (ABCC9) and potassium inwardly rectifying channelsubfamily J member 8 (KCNJ8).
 7. The cells of claim 1, wherein the cellsare capable of inducing pericyte-driven phenomena in bone marrowmicrovascular endothelial cells (BMECs), including the enhancement ofbarrier properties and reduction of transcytosis. 8.-12. (canceled) 13.A population of NCSC created by (a) culturing hPSC in E6-CSFD medium forabout 15 days to produce p75-NGFR+HNK+NCSC cells, (b) sorting p75-NGFR⁺cells and re-plating the p75-NGFR⁺ cells to produce an enrichedpopulation of p75-NGFR⁺NCSCs.
 14. A population of brain pericyte-likecells wherein the cells express pericyte markers but do not expressACTA2 and wherein the cells are generated from human pluripotent stemcells (hPSC), comprising the steps of c. culturing the cells of step (b)of claim 13 in E6 media with an addition of serum for about 11 days,wherein a brain pericyte-like population of cells that express pericytemarkers but do not express ACTA2 is produced.
 15. An isogenicblood-brain barrier (BBB) model created using the cells of claim
 14. 16.A method of creating a population of p75-NGFR+HNK+NCSCs from humanpluripotent stem cells, the method comprising: a. culturing hPSC inE6-CSFD medium for about 15 days to produce p75-NGFR+HNK+NCSCs, and b.sorting p75-NGFR⁺ cells and re-plating the p75-NGFR⁺ cells of step (a)to produce a population of p75-NGFR⁺NCSCs.
 17. The method of claim 16,wherein step (b) is performed by magnetic activated cell sorting. 18.The method of claim 16, wherein the population of cells produced isp′75-NGFR⁺HNK⁺AP-2⁺NCSCs which are able to double at least 5 times inculture and maintain expression p75-NGFR⁺, HNK⁺, and AP-2⁺ within thecells.
 19. The method of claim 16, wherein the NCSCs are able to bemaintained in culture for at least five passages and maintainNGFR⁺HNK⁺AP-2⁺ marker expression and do not express pericyte markers.20. The method of claim 16, the method further comprising c. culturingthe cells of step (b) in E6 media with an addition of serum for about 11days, wherein the NCSCs produce a population of a brain pericyte-likecells that express NG2, and PDGFRB but do not express ACTA2 is produced.21. NCSCs produced by the method of claim 16, wherein the NCSCs maintainthe potential to differentiate into neurons and mesenchymal cells.